BDNF对小鼠未成熟卵母细胞的作用及机制研究
|
摘要
第一部分BDNF对小鼠未成熟卵母细胞体外成熟及胚胎发育能力的影响
目的：观察BDNF对小鼠未成熟卵母细胞的体外成熟及胚胎发育能力的影响，并选择BDNF作用于小鼠未成熟卵母细胞的最适合浓度。
方法：实验分三个部分。首先在常规体外成熟培养液(以α-MEM为基础培养液，添加10％的FBS和0．2IU/ml的卵泡刺激素FSH)中，添加不同浓度(0、1ng/ml、5ng/ml、10ng/ml)的BDNF，培养小鼠卵丘-卵母细胞复合物(CEOs)，成熟后进行体外受精，观察卵母细胞成熟率、受精率和囊胚形成率，从中选择BDNF作用于小鼠未成熟卵的最佳浓度(5ng/ml)；其次，在常规体外成熟培养液中除去FSH条件下，改变FBS的浓度(0、5％、10％)，添加BDNF(0、5ng/ml)，观察不同FBS条件下，BDNF对小鼠CEOs体外成熟及发育能力的影响，明确BDNF与FBS对小鼠未成熟卵作用的相互关系。最后将CEOs脱去卵丘细胞，用添加5％的FBS的体外成熟培养液培养，观察BDNF5ng/ml的添加与否对裸卵体外成熟及发育能力的影响，从而了解BDNF对未成熟卵的作用是否需要卵丘细胞的介导。
结果：当体外成熟培养液中含有FSH和10％的FBS时，与体外成熟对照组比较，BDNF各浓度组卵母细胞的成熟率和受精率差异均无统计学意义，但BDNF5ng/ml组的囊胚形成率(75．00%)显著高于体外成熟对照组(56．63%)，而接近体内成熟组(76．92％)。因此，BDNF5ng/ml是促进未成熟卵成熟和囊胚发育的最佳浓度；当培养液中仅含FBS(5％或10％)时，与相应的对照组比较，卵母细胞成熟率和受精率差异无统计学意义，但BDNF显著提高囊胚形成率(FBS5％：21．51％vs．9．09%；FBS10％：31．76％vs．18．9％)；当培养液中不含FBS、FSH时，虽然两组均无囊胚形成，但BDNF显著提高了卵母细胞的受精率(29．72% vs．10．57％)。同样，当BDNF作用于未成熟裸卵时，虽然不影响成熟率和受精率，但将囊胚形成率提高了3倍(18．18％vs．4．65％)。
结论：BDNF作用于小鼠未成熟卵母细胞的最适合浓度为5ng/ml。在不同的体外成熟培养条件下，BDNF不影响卵母细胞成熟率，也无须卵丘细胞的介导，直接作用于卵母细胞，促进小鼠卵母细胞质的发育，提高卵母细胞的发育能力。
第二部分BDNF对体外成熟的卵母细胞纺锤体形态；和皮质颗粒分布的影响目的：纺锤体和皮质颗粒是卵母细胞内的重要细胞器，纺锤体的形态、大小、位置以及皮质颗粒的分布都常用作评价卵母细胞质量的指标。在小鼠未成熟卵体外培养过程中，研究BDNF对纺锤体形态、大小、位置以及皮质颗粒分布的影响，了解BDNF在细胞水平对卵母细胞作用的具体环节。方法：将卵丘-卵母细胞复合物分别培养在两种α-MEM体外成熟培养液中(均含5％FBS，不添加或添加5ng/mlBDNF)，收集体外培养2h、4h、8h、12h、16h的卵母细胞，同时取HCG注射后2h、4h、8h、12h、16h的体内成熟卵母细胞作为正常对照。用免疫荧光法标记微管和中心粒周蛋白，Hochest33258标记染色质，FITC-LCA标记皮质颗粒，共聚焦显微镜下分别观察卵母细胞减数分裂进程、纺锤体形态和皮质颗粒分布，并进行比较。结果：体内和体外成熟的卵母细胞在减数分裂进程、纺锤体形态和皮质颗粒分布上表现出显著的差异。与体外成熟卵母细胞比较，体内成熟的卵母细胞减数分裂过程表现出高度的同步性。BDNF不影响卵母细胞IVM2h减数分裂的恢复和IVM16h的成熟率，但影响了减数分裂的中间过程。在IVM8h和12h，BDNF处理组和对照组卵母细胞所处的减数分裂阶段存在明显的差异。体内成熟卵母细胞减数分裂中期(包括MⅠ和MⅡ)的纺锤体呈两端逐渐变细的“纺锤状”，微管排列紧密，胞质中存在较多的微管形成中心，纺锤体100%靠近卵膜；体外成熟的卵母细胞的纺锤体两端宽大，呈“桶状”，微管排列稀疏，胞质中的微管形成中心较少，纺锤体靠近卵膜的比例明显降低。在体外成熟培养液中添加BDNF的条件下，卵母细胞MⅠ纺锤体的宽度和表面积比对照组明显减少(P＜0．05)；微管形成中心明显增多(P＜0．01)，MⅠ和MⅡ期纺锤体靠近卵膜的比例明显增加(MⅠ：65．79% vs．34．29；MⅡ：71．939% vs．43．24%。P＜0．01)。卵母细胞体内和体外成熟过程中，皮质颗粒重排的时机不同，体内成熟卵母细胞在皮质颗粒分布方面同样表现出高度的一致性。而在卵母细胞体外成熟的BDNF处理组和对照组中，尽管IVM4h和16h皮质颗粒分布相似，但在IVM8h和12h，皮质颗粒分布的类型在统计学上存在明显的差异(P＜0．05)。IVM8h，BDNF处理组出现第一次无皮质颗粒区(StageⅣ)的卵母细胞(31．25％)明显多于对照组(12．50%)；IVM12h，BDNF处理组出现皮质颗粒在第一极体排出时形成的分裂沟中聚集(StageⅤ)的卵母细胞多于对照组，但对照组出现第二次无皮质颗粒区(StageⅥ)的卵母细胞多于BDNF处理组。
结论：BDNF在一定程度上改变了卵母细胞减数分裂进程，纺锤体的形态、定位以及皮质颗粒的重排。BDNF对减数分裂中期纺锤体形态和定位的改善可能是其促进卵母细胞质成熟的一个重要原因。
第三部分BDNF作用于体外成熟的卵母细胞的信号转导途径
目的：通过检测体外成熟过程中卵母细胞和卵丘细胞内蛋白激酶B(PKB)和丝裂原活化蛋白激酶(MAPK)的磷酸化的变化，来了解BDNF对PKB和MAPK途径的影响，初步探索BDNF促进卵母细胞成熟的信号转导途径。
方法：将卵丘-卵母细胞复合物分为三个组：α-MEM中添加5％的FBS(对照组)；α-MEM中添加5％的FBS和5ng/mlBDNF(BDNF处理组)；α-MEM中添加5％的FBS、5ng/mlBDNF和100nMK252a(一种Trk受体抑制剂，K252a处理组)。分别进行体外培养，在培养0h、1h、2h、3h、6h、16h收集卵母细胞和卵丘细胞。用Western Blot方法检测卵母细胞和卵丘细胞内PKB和MAPK的磷酸化变化并进行比较。
结果：在卵母细胞内BDNF增强了PKB的活性，延长了PKB的激活时间。而在Trk受体抑制剂K252a的作用下，PKB的活性被完全抑制。因此，BDNF对卵母细胞内PKB的激活是通过与受体TrkB结合后产生的效应。BDNF也在一定程度上增强了MAPK的活性，然而并不是TrkB的受体后效应，因为K252a不能抑制MAPK的激活。在卵丘细胞内，BDNF延长了PKB和MAPK活性持续的时间。K252a能短暂地抑制了卵丘细胞内PKB的活性，但对MAPK的活性无影响。IVM16h，对照组卵丘细胞内PKB和MAPK的蛋白总量下降，BDNF处理组则保持PKB和MAPK的蛋白总量不变。
结论：BDNF增强了卵母细胞内PKB和MAPK的活性，延长了卵丘细胞内PKB和MAPK的激活时间。而这可能与BDNF改善体外成熟的卵母细胞质的成熟有关。在卵母细胞，PKB途径是BDNF与受体TrkB结合的信号转导途径之一，而MAPK途径则不是BDNF直接激活的信号转导途径。
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 competence
Objective 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 vitro
Objective 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 oocytes
Objective 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.
目的：观察BDNF对小鼠未成熟卵母细胞的体外成熟及胚胎发育能力的影响，并选择BDNF作用于小鼠未成熟卵母细胞的最适合浓度。
方法：实验分三个部分。首先在常规体外成熟培养液(以α-MEM为基础培养液，添加10％的FBS和0．2IU/ml的卵泡刺激素FSH)中，添加不同浓度(0、1ng/ml、5ng/ml、10ng/ml)的BDNF，培养小鼠卵丘-卵母细胞复合物(CEOs)，成熟后进行体外受精，观察卵母细胞成熟率、受精率和囊胚形成率，从中选择BDNF作用于小鼠未成熟卵的最佳浓度(5ng/ml)；其次，在常规体外成熟培养液中除去FSH条件下，改变FBS的浓度(0、5％、10％)，添加BDNF(0、5ng/ml)，观察不同FBS条件下，BDNF对小鼠CEOs体外成熟及发育能力的影响，明确BDNF与FBS对小鼠未成熟卵作用的相互关系。最后将CEOs脱去卵丘细胞，用添加5％的FBS的体外成熟培养液培养，观察BDNF5ng/ml的添加与否对裸卵体外成熟及发育能力的影响，从而了解BDNF对未成熟卵的作用是否需要卵丘细胞的介导。
结果：当体外成熟培养液中含有FSH和10％的FBS时，与体外成熟对照组比较，BDNF各浓度组卵母细胞的成熟率和受精率差异均无统计学意义，但BDNF5ng/ml组的囊胚形成率(75．00%)显著高于体外成熟对照组(56．63%)，而接近体内成熟组(76．92％)。因此，BDNF5ng/ml是促进未成熟卵成熟和囊胚发育的最佳浓度；当培养液中仅含FBS(5％或10％)时，与相应的对照组比较，卵母细胞成熟率和受精率差异无统计学意义，但BDNF显著提高囊胚形成率(FBS5％：21．51％vs．9．09%；FBS10％：31．76％vs．18．9％)；当培养液中不含FBS、FSH时，虽然两组均无囊胚形成，但BDNF显著提高了卵母细胞的受精率(29．72% vs．10．57％)。同样，当BDNF作用于未成熟裸卵时，虽然不影响成熟率和受精率，但将囊胚形成率提高了3倍(18．18％vs．4．65％)。
结论：BDNF作用于小鼠未成熟卵母细胞的最适合浓度为5ng/ml。在不同的体外成熟培养条件下，BDNF不影响卵母细胞成熟率，也无须卵丘细胞的介导，直接作用于卵母细胞，促进小鼠卵母细胞质的发育，提高卵母细胞的发育能力。
第二部分BDNF对体外成熟的卵母细胞纺锤体形态；和皮质颗粒分布的影响目的：纺锤体和皮质颗粒是卵母细胞内的重要细胞器，纺锤体的形态、大小、位置以及皮质颗粒的分布都常用作评价卵母细胞质量的指标。在小鼠未成熟卵体外培养过程中，研究BDNF对纺锤体形态、大小、位置以及皮质颗粒分布的影响，了解BDNF在细胞水平对卵母细胞作用的具体环节。方法：将卵丘-卵母细胞复合物分别培养在两种α-MEM体外成熟培养液中(均含5％FBS，不添加或添加5ng/mlBDNF)，收集体外培养2h、4h、8h、12h、16h的卵母细胞，同时取HCG注射后2h、4h、8h、12h、16h的体内成熟卵母细胞作为正常对照。用免疫荧光法标记微管和中心粒周蛋白，Hochest33258标记染色质，FITC-LCA标记皮质颗粒，共聚焦显微镜下分别观察卵母细胞减数分裂进程、纺锤体形态和皮质颗粒分布，并进行比较。结果：体内和体外成熟的卵母细胞在减数分裂进程、纺锤体形态和皮质颗粒分布上表现出显著的差异。与体外成熟卵母细胞比较，体内成熟的卵母细胞减数分裂过程表现出高度的同步性。BDNF不影响卵母细胞IVM2h减数分裂的恢复和IVM16h的成熟率，但影响了减数分裂的中间过程。在IVM8h和12h，BDNF处理组和对照组卵母细胞所处的减数分裂阶段存在明显的差异。体内成熟卵母细胞减数分裂中期(包括MⅠ和MⅡ)的纺锤体呈两端逐渐变细的“纺锤状”，微管排列紧密，胞质中存在较多的微管形成中心，纺锤体100%靠近卵膜；体外成熟的卵母细胞的纺锤体两端宽大，呈“桶状”，微管排列稀疏，胞质中的微管形成中心较少，纺锤体靠近卵膜的比例明显降低。在体外成熟培养液中添加BDNF的条件下，卵母细胞MⅠ纺锤体的宽度和表面积比对照组明显减少(P＜0．05)；微管形成中心明显增多(P＜0．01)，MⅠ和MⅡ期纺锤体靠近卵膜的比例明显增加(MⅠ：65．79% vs．34．29；MⅡ：71．939% vs．43．24%。P＜0．01)。卵母细胞体内和体外成熟过程中，皮质颗粒重排的时机不同，体内成熟卵母细胞在皮质颗粒分布方面同样表现出高度的一致性。而在卵母细胞体外成熟的BDNF处理组和对照组中，尽管IVM4h和16h皮质颗粒分布相似，但在IVM8h和12h，皮质颗粒分布的类型在统计学上存在明显的差异(P＜0．05)。IVM8h，BDNF处理组出现第一次无皮质颗粒区(StageⅣ)的卵母细胞(31．25％)明显多于对照组(12．50%)；IVM12h，BDNF处理组出现皮质颗粒在第一极体排出时形成的分裂沟中聚集(StageⅤ)的卵母细胞多于对照组，但对照组出现第二次无皮质颗粒区(StageⅥ)的卵母细胞多于BDNF处理组。
结论：BDNF在一定程度上改变了卵母细胞减数分裂进程，纺锤体的形态、定位以及皮质颗粒的重排。BDNF对减数分裂中期纺锤体形态和定位的改善可能是其促进卵母细胞质成熟的一个重要原因。
第三部分BDNF作用于体外成熟的卵母细胞的信号转导途径
目的：通过检测体外成熟过程中卵母细胞和卵丘细胞内蛋白激酶B(PKB)和丝裂原活化蛋白激酶(MAPK)的磷酸化的变化，来了解BDNF对PKB和MAPK途径的影响，初步探索BDNF促进卵母细胞成熟的信号转导途径。
方法：将卵丘-卵母细胞复合物分为三个组：α-MEM中添加5％的FBS(对照组)；α-MEM中添加5％的FBS和5ng/mlBDNF(BDNF处理组)；α-MEM中添加5％的FBS、5ng/mlBDNF和100nMK252a(一种Trk受体抑制剂，K252a处理组)。分别进行体外培养，在培养0h、1h、2h、3h、6h、16h收集卵母细胞和卵丘细胞。用Western Blot方法检测卵母细胞和卵丘细胞内PKB和MAPK的磷酸化变化并进行比较。
结果：在卵母细胞内BDNF增强了PKB的活性，延长了PKB的激活时间。而在Trk受体抑制剂K252a的作用下，PKB的活性被完全抑制。因此，BDNF对卵母细胞内PKB的激活是通过与受体TrkB结合后产生的效应。BDNF也在一定程度上增强了MAPK的活性，然而并不是TrkB的受体后效应，因为K252a不能抑制MAPK的激活。在卵丘细胞内，BDNF延长了PKB和MAPK活性持续的时间。K252a能短暂地抑制了卵丘细胞内PKB的活性，但对MAPK的活性无影响。IVM16h，对照组卵丘细胞内PKB和MAPK的蛋白总量下降，BDNF处理组则保持PKB和MAPK的蛋白总量不变。
结论：BDNF增强了卵母细胞内PKB和MAPK的活性，延长了卵丘细胞内PKB和MAPK的激活时间。而这可能与BDNF改善体外成熟的卵母细胞质的成熟有关。在卵母细胞，PKB途径是BDNF与受体TrkB结合的信号转导途径之一，而MAPK途径则不是BDNF直接激活的信号转导途径。
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 competence
Objective 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 vitro
Objective 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 oocytes
Objective 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.
引文
1．Gilchrist RB， Thompson JG. Oocyte maturation： emerging concepts and technologies to improve developmental potential in vitro．Theriogenology， 2007， 67：6-15．
2．Park JY， Su YQ， Ariga M， et al．EGF-like growth factors as midiators of LH action in the ovulatory follicle．Science， 2004，303 (5658)：682-684．
3．Kawamura K， Kumagai J， Sudo S et al．Paracrine regulation of mammalian oocyte maturation and male germ cell survival．Proc Natl Acad Sci U S A， 2004．101(19)：7323-7328．
4．Ofeda SR， Romero C， Tapia C， et al．Neurotrophic and cell-cell dependent control of early follicular development．Mol cell Endocrinol， 2001，63：67-71．
5．Harel S， Jin S．Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod， 2006， 12(6)：357-365．
6．Paredes A， Romero C，Dissen GA， et al．TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary．Dev Biol， 2004， 267： 430-449．
7．Seifer DB， Feng B， Shelden RM．et al．Brain-derived neurotrophic factor： a novel human ovarian follicular protein．J Clin Endocrinol Metab， 2002， 87(2)： 655-659．
8．Seifer DB， Feng B， Shelden RM．Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol．2006， 194(4)： 1129-1134．
9．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
10．Kawamura K．Kawamura N， Mulders SM， et al．Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos．Proc Natl Acad Sci U S A， 2005， 102(26)：9206-9211．
11．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development．Reproduction， 2005，129 (4)：423-434
12．Lee E， Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
13．Sirard MA， Richard F， Blondin P， et al．Contribution of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
14．Rose CR， Blum R， Kafitz KW， et al．From modulator to mediator： rapid effects of BDNF on ion channels．Bio Essays， 2004， 26：1185-1194．
15．Fan HY， Sun QY Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals．Biol reprod， 2004， 70： 535-547．
16．Andersen CB， Sakaue H， Nedachi T，et al．Protein kinase B/Akt is essential for the insulin-but not progesterone-stimulated resumption of meiosis in Xenopus oocytes．Biochem J， 2003， 369(Pt 2)：227-238．
17．Hoshino Y， Sato E．Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes．Dev Biol， 2008， 314： 215-223．
18．Tosti E．Calcium ion currents mediating oocyte maturation events．Reprod Biol Endocrinol， 2006，4：26-37．
1．Jensen T， Johnson AL．Expression and function of brain-derived neurotrophin factor and its receptor， TrkB．in ovarian follicles from the domestic hen (Gallus gallus domesticus)．J Exp Biol， 2001，204(Pt 12)： 2087-2095．
2．Kawamura K， Kawamura N．Mulders SM， et al．Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos．Proc Natl Acad Sci U S A．2005， 102(26)： 9206-9211．
3．Dissen GA， Hirshfield AN， Malamed S， et al．Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated： changes at the time of folliculogenesis．Endocrinology， 1995， 136(10)： 4681-4692．
4．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotesbovineoocytecytoplasmiccompetenceforembryodevelopment．Reproduction， 2005， 129(4)： 423-434．
5．Lee E．Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
6．Seifer DB， Feng B， Shelden RM．Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol， 2006．194(4)： 1129-1134．
7．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
8．Harel S， Jin S， Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod， 2006， 12(6)： 357-365．
9．Seifer DB， Feng B， Shelden RM， et al．Brain-derived neurotrophic factor： a novel human ovarian follicular protein．J Clin Endocrinol Metab， 2002， 87(2)： 655-659．
10．Paredes A， Romero C， Dissen GA， et al．TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary．Dev Biol， 2004， 267 (2)：430-449．
11．Ofeda SR， Romero C， Tapia C， et al．Neurotrophic and cell-cell dependent control of early follicular development．Mol cell Endoorinol， 2000， 163(1-2)：67-71．
12．Goud PT， Goud AP， Qian C，．et al．In-vitro maturation of human germinal vesicle stage oocytes： role of cumulus cells and epidermal growth factor in the culture medium．Hum．Reprod， 1998， 13(6)： 1638-1644．
13．Demeestere Ⅰ， Gervy C， Centner J．et al．Effect of insulin-like growth factor-Ⅰ during preantral follicular culture on steroidogenesis， in vitro oocyte maturation， and embryo development in mice．Biol Reprod， 2004， 70(6)： 1664-1669．
14．Stock AE， Woodruff TK， Smith LC．Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone-and serum-free medium．Biol Reprod， 1997，56： 1559-1564．
15．Ikeda S．Ichihara-Tanaka K， Azuma T．et al．Effects of midkine during in vitro maturation of bovine oocytes on subsequent developmental competence．Biol Reprod，2000， 63：1067-1074．
1．Sirard MA， Richard F， Blondin P， et al．Contribution．，of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
2．Liu H， Krey LC， Zhang J， et al．Ooplasmic influence on nuclear function during the metaphase Ⅱ-interphase transition in mouse oocytes．Biol Reprod， 2001，65：1794-1799．
3．Salamone DF， Damiani P， Fissore RA， et al．Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised．Biol Reprod，2001，64： 1761-1768．
4．Hu Y．Betzendahl I， Cortvrindt R， et al．Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture．Hum Reprod， 2001，16：737-748．
5．Eppig JJ， O'Brien MJ．Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo．Theriogenology， 1998， 49：415-422．
6．Mermillod P， Oussaid B， Cognie Y Aspects of follicular and oocyte maturation that affect the developmental potential of embryos．J Reprod Fertil Suppl， 1999， 54：449-460．
7．Sanfins A．Lee GY， Plancha CE， et al．Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes．Biol Reprod．2003，69： 2059-2067．
8．Liu XY， Mal SF， Miao DQ， et al．Cortical granules behave differently in mouse oocytes matured under different conditions．Hum reprod．2005， 20： 3402-3413．
9．Ferreira EM， Vireque AA， Adona PR， et al．Cytoplasmic maturation of bovine oocytes：Structural and biochemical modifications and acquisition of developmental competence．Theriogenology， 2009．71 (5)： 836-848．
10．Wang Q， Sun QYEvaluation of oocyte quality： morphological， cellular and molecular predictors．Reprod Fertil Dev， 2007， 19(1)： 1-12．
11．Sun QY， Wu GM， Lai L， et al．Translocation of active mitochondria during pig oocyte maturation， fertilization and early embryo development in vitro．Reproduction， 2001， 122： 155-163．
12．Combelles CM， Racowsky C．Assessment and optimization of oocyte quality during assisted reproductive technology treatment．Semin Reprod Med， 2005， 23：277-284．
13．Sanfins A， Plancha CE， Overstrom EW， et al．Meiotic spindle morphogenesis in in vivo and in vitro matured mouse oocytes： insights into the relationship between nuclear and cytoplasmic quality．Hum Reprod， 2004， 19(12)： 2889-2899．
14．Gilchrist RB， Thompson JG．Oocyte maturation： Emerging concepts and technologies to improve developmental potential in vitro．Theriogenology， 2007， 67： 6-15．
15．张玲，朱桂金．丁内酯-Ⅰ对小鼠卵母细胞体外成熟及发育能力的影响．生殖与避孕，2005， 25 (7)： 396-399．
16．Combelles CM， Albertini DF．Microtubule patterning during meiotic maturation in mouse oocytes is determined by cell cycle-specific sorting and redistribution of gamma-tubulin．Dev Biol．2001，239： 281-294．
17．Patrizio P， Fragouli E， Bianchi V，et al．Molecular methods for selection of the ideal oocyte．Reprod Biomed Online， 2007， 15(3)： 346-353．
18．Leader B， Lim H， Carabatsos MJ， et al．Formin-2， polyploidy．hypofertility and positioning of the meiotic spindle in mouse oocytes．Nat Cell Biol， 2002， 4： 921-928．
19．Sun QY， Schatten H．Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction, 2006. 13: 193-205.
20. Tapia-Arancibia L, Rage F, Givalois L, et al. Physiology of BDNF: focus on hypothalamic function. Front Neuroendocrinol, 2004, 25(2): 77-107.
21. Rose CR, Blum R, Kafitz Kw, Kovalchuk Y, Konnerth A. From modulator to mediator: rapid effects of BDNF on ion channels. BioEssays, 2004, 26: 1185-1194.
22. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol reprod, 2004, 70: 535-547.
23. Andersen CB, Sakaue H, Nedachi T, et al. Protein kinase B/Akt is essential for the insulin-but not progestone-stimulated resumption of meiosis in Xenopus oocytes. BioChem J, 2003, 369: 227-238.
24. Hoshino Y, Sato E. Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes. Dev Biol, 2008, 314: 215-223.
25. Han SJ, Vaccari S, Nedachi T, et al. Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation. EMBO J, 2006, 25(24): 5716-5725.
26. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005, 281: 160-170.
27. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
28. Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179:25-34.
1. Friedman WJ, Greene LA. Neurotrophin Signaling via Trks and p75. Exp Cell Res, 1999,253: 131-142.
2. Tapia-Arancibia L, Rage F, Givalois L, et al. Physiology of BDNF: focus on hypothalamic function. Front Neuroendocrinol, 2004, 25(2): 77-107.
3. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol reprod 2004; 70: 535-547.
4. Su YQ, Wigglesworth K, Pendola FL, et al. Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology, 2002, 143: 2221-2232.
5. Hoshino Y, Sato E. Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes. Dev Biol, 2008, 314: 215-223.
6. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005/281: 160-170.
7. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
8. Shimada M, Ito J, Yamashita Y, et al. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179: 25-34.
9. Andersen CB, Sakaue H, Nedachi T, et al. Protein kinase B/Akt is essential for the insulin-but not progestone-stimulated resumption of meiosis in Xenopus oocytes. BioChem J, 2003, 369: 227-238.
10. Tosti E. Calcium ion currents mediating oocyte maturation events. Reprod Biol Endocrinol, 2006, 4: 26-37.
11. Ofeda SR, Romero C, Tapia C, et al. Neurotrophic and cell-cell dependent control of early follicular development. Mol cell Endoorinol, 2000, 163 (1-2) : 67-71.
12. Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J, 1999, 18(3): 616-622.
13. Kawamura K, Kawamura N, Mulders SM, et al. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci U S A, 2005, 102(26): 9206-9211.
14. Lengyel F, Vertes Z, Kovacs KA,et al. Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus. J. Steroid. Biochem. Mol Biol, 2004, 91, 285-288.
15. Viniegra JG, Martinez N, Modirassari P, et al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem, 2005, 280: 4029-4036.
16. Kalous J, Solc P, Baran(?) V, et al. PKB/AKT is involved in resumption of meiosis in mouse oocytes. Biol cell, 2006, 98, 111-123.
17. Patrizio P, Fragouli E, Bianchi V, et al. Molecular methods for selection of the ideal oocyte. Reprod Biomed Online, 2007, 15(3): 346-353.
18. Su YQ, Wigglesworth K, Pendola FL, et al. Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology, 2002, 143: 2221-2232.
19. Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem, 2003, 72: 609-642.
1．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotesbovineoocytecytoplasmiccompetenceforembryodevelopment．Reproduction， 2005， 129(4)： 423-434．
2．Tapia-Arancibia L， Rage F， Givalois L．et al．Physiology of BDNF： focus on hypothalamic function．Front Neuroendocrinol，2004， 25(2)： 77-107．
3．付娟，邢福棋．脑源性神经营养因子与卵巢．国外医学计划生育分册，2003， 22(4)： 207-210．
4．Lee R， Kermani P．Teng KK， et al．Regulation of cell survival by secreted proneurotrophins．Science， 2001，294： 1945-1948．
5. Ofeda SR, Romero C, Tapia C, et al. Neurotrophic and cell-cell dependent control of early follicular development. Mol cell Endoorinol, 2000, 163 (1-2) : 67-71.
6. Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J, 1999, 18(3): 616-622.
7. Mischel PS, Smith SG, Vining ER,et al. The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA. J Biol Chem, 2001, 276(14): 11294-11301.
8. Eide FF, Vining ER, Eide BL,et al. Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J Neurosci, 1996, 16 (10): 3123-3129.
9. Paredes A, Romero C, Dissen GA, et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary. Dev Biol, 2004, 267 (2) : 430-449
10. Kerr B, Garcia-Rudaz C, Dorfman M, et al. TrkA and TrkB receptors facilitate follicle assembly and early follicular development in the mouse ovary. Reproduction, 2009. [Epub ahead of print]
11. Dissen GA, Hirshfield AN, Malamed S, et al. Expression of:heurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis. Endocrinology, 1995, 136(10): 4681-4692.
12. Dissen GA, Garcia-Rudaz C, Ojeda SR. Role of neurotrophic factors in early ovarian development. Semin Reprod Med, 2009, 27(1): 24-31.
13. Kawamura K, Kawamura N, Mulders SM, et al. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci U S A, 2005, 102(26): 9206-9211.
14. Seifer DB, Feng B, Shelden RM, et al. Brain-derived neurotrophic factor: a novel human ovarian follicular protein. J Clin Endocrinol Metab, 2002, 87(2): 655-659.
15. Seifer DB, Lambert-Messerlian G, Schneyer AL. Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women. Fertil Steril, 2003, 79: 451-452.
16. Seifer DB, Feng B, Shelden RM. Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol， 2006．194(4)： 1129-1134．
17．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
18．Harel S， Jin S， Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod．2006， 12(6)：357-365．
19．Lee E， Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
20．Jensen T， Johnson AL．Expression and function of brain-derived neurotrophin factor and its receptor， TrkB， in ovarian follicles from the domestic hen (Gallus gallus domesticus)．J Exp Biol， 2001，204(Pt 12)： 2087-2095
21．付娟，邢福祺，陈士岭等．促性腺激素对人卵巢脑源性神经营养因子及其受体表达的影响．生殖与避孕，2003，23 (3)：149-152．
22．Begliuomini S， Casarosa E， Pluchino N．et al．Influence of endogenous and exogenous sex hormones on plasma brain-derived neurotrophic factor．Hum Reprod， 2007， 22(4)：995-1002．
23．Monteleone P， Artini PG，SimiG，et al．Brain derived neurotrophic factor circulating levels in patients undergoing IVF．J Assist Reprod Genet， 2007， 24： 477-480．
24．Piccinni A， Marazziti D， Del Debbio A， et al．Diurnal variation of plasma brain-derived neurotrophic factor (BDNF) in humans： an analysis of sex differences．Chronobiol Int，2008．25(5)： 819-826．
25．Huang EJ．Reichardt LF．Trk receptors： roles in neuronal signal transduction．Annu Rev Biochem．2003， 72： 609-642．
26．Fan HY， Sun QY．Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals．Biol reprod， 2004， 70： 535-547．
27．Su YQ， Wigglesworth K， Pendola FL， et al．Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse．Endocrinology， 2002， 143：2221-2232．
28．Hoshino Y， Sato E．Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes．Dev Biol， 2008， 314： 215-223．
29. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005, 281: 160-170.
30. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
31. Shimada M, Ito J, Yamashita Y, et al. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179: 25-34.
32. Tosti E. Calcium ion currents mediating oocyte maturation events. Reprod Biol Endocrinol, 2006, 4: 26-37.
33. Park C, Choi WS, Kwon H, et al. Temporal and spatial expression of neurotrophins and their receptors during male germ cell development. Mol Cells, 2001, 12(3):360-367.
34. Wheeler EF, Bothwell M. Spatiotemporal patterns of expression of NGF and the low-affinity NGF receptor in rat embryos suggest functional, roles in tissue morphogenesis and myogenesis. JNeurosci, 1992, 12(3): 930-945.
35. Muller D, Davidoff MS, Bargheer O, et al. The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells. Histochem Cell Biol, 2006, 126(2): 199-211.
1．Sirard MA．Richard F， Blondin P， et al．Contribution of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
2．Ferreira EM， Vireque AA， Adona PR， et al．Cytoplasmic maturation of bovine oocytes： Structural and biochemical modifications and acquisition of developmental competence．Theriogenology， 2009．71 (5)： 836-848．
3．Wang Q， Sun QYEvaluation of oocyte quality： morphological， cellular and molecular predictors．Reprod Fertil Dev， 2007， 19(1)： 1-12．
4．Krisher R L．The effect of oocyte quality on development．J Anim Sci， 2004，82： E14-E23.
5. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryoquality. Hum Reprod, 1997, 12(8): 1750-1755.
6. Ten J, Mendiola J, Vioque J, et al. Donor oocyte dysmorphisms and their influence on fertilization and embryo quality. Reprod Biomed Online, 2007, 14(1): 40-48.
7. Kahraman S, Yakin K, Donmez E, et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum. Reprod, 2000,15:2390-2393.
8. Balaban B, Urman B. Effect of oocyte morphology on embryo development and implantation.Reprod Biomed Online, 2006, 12(5):608-615.
9. Gabrielsen, A, Lindenberg, S, Petersen, K. The impact of the zona pellucida thickness variation of human embryos on pregnancy outcome in relation to suboptimal embryo development. A prospective randomized controlled study. Hum Reprod, 2001, 16: 2166-2170.
10. Hu Y, Betzendahl I, Cortvrindt R, et al. Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. Hum Reprod , 2001, 16: 737-748.
11. Mermillod P, Oussaid B, Cognie Y. Aspects of follicular and oocyte maturation that affect the developmental potential of embryos. J Reprod Fertil Suppl, 1999, 54: 449-460.
12. Cooke S, Tyler J P, Driscoll G L. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod, 2003, 18: 2397-2405.
13. Sanfins A, Lee GY, Plancha CE, et al. Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes. Biol Reprod, 2003, 69: 2059-2067.
14. Rossi G, Macchiarelli G, Palmerini MG, et al. Meiotic spindle configuration is differentially influenced by FSH and epidermal growth factor during in vitro maturation of mouse oocytes. Hum Reprod, 2006, 21: 1765-1770.
15. Moon J H, Hyun C S, Lee S W, et al. Visualization of the metaphase Ⅱ meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum. Reprod, 2003, 18: 817-820.
16. Wang WH, Meng L, Hackett R J, et al. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod, 2001,16: 1464-1468.
17. De Santis L, Cino I, Rabellotti E, et al. Polar body morphology and spindle imaging as predictors of oocyte quality. Reprod Biomed Online, 2005, 11(1): 36-42.
18. Lonergan P, Monaghan P, Rizos D, et al. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mo. Reprod Dev, 1994, 37: 48-53.
19. Borini A, Maccolini A, Tallarini A, et al. PerifoUicular vaseularity and its relationship with oceyte maturity and IVF outcome. Ann N Y Acad Sci, 2001, 943: 64-67.
20. Wilding M, Dale B, Marino M, et al. Mitochondrial aggregation.patterns and activity in human oocytes and preimplantation embryos. Hum Reprod, 2001,16: 909-917.
21. Singh R, Sinclair KD. Metabolomics: approaches to assessing:* iooeyte and embryo quality.Theriogenology, 2007, 68 Suppl 1: S56-62.
22. Bavister BD, Squirrell JM. Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod, 2000, 15: 189-198.
23. Ludwig M, Schopper B, AI-Hasani S, et al. Clinical use of a pronuclear stage score following intracytoplasmic sperm injection: impact on pregnancy rates under the condition of the German embryo protection law. Hum Reprod, 2000, 15(2): 325-329.
24. Brevini T A, Vassena R, Francisci C, et al. Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol Reprod, 2005, 72: 1218-1223.
25. Keefe D, Niven-Fairchild T, Powel S, et al .Mitochondrial deoxyribonucleic acid deletions in oocytes of reproductively aging women. Fertil Steril, 1995, 64(3): 577-583.
26. HsiehRH, Au HK, Yeh TS, et al. Decreased expression of mitochondrial genes in human unfertilized oocytes and arrested embryos. Fertil Steril, 2004, 81 [Suppl 1]: 912-918.
27. Van Blerkom J. Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction, 2004, 128(3): 269-280.
28. Santos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril, 2006, 85: 584-591.
29. Stojkovic M, Machado SA, Stojkovic P, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod, 2001, 64: 904-909.
30. Tamassia M, Nuttinck F, May-Panloup P, et al. In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup. Biol Reprod, 2004, 71: 697-704.
31. Liu XY, Mal SF, Miao DQ, et al. Cortical granules behave differently in mouse oocytes matured under different conditions. Hum reprod, 2005, 20: 3402-3413.
32. Ferrandi B, Cremonesti G, Geiger R, et al. Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation. Acta Histochem, 1993, 95: 89-96.
33. Ericsson SA, Boice ML, Funahashi H, et al. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology, 1993, 39: 214.
34. Alm H, Torner H, Lohrke B, et al. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresyl blue staining before IVM as indicator for glucose-6- phosphate dehydrogenase activity. Theriogenology, 2005, 63: 2194-2205.
35. El Shourbagy SH, Spikings EC, Freitas M, et al. Mitochondria directly influence fertilization outcome in the pig. Reproduction, 2006, 131: 233-245.
36. Baart EB, van den Berg I, Martini E, et al. FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos: the added value of extended aneuploidy detection. Prenatal Diagnosi, 2007, 27: 55-63.
37. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod, 2000, 6: 1055-1062.
38. Munne S, Chen S, Fischer J et al. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertil Steril, 2005, 84: 331-335.
39. Kuliev A, Cieslak J, Ilkevitch Y, et al. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod BioMed Online, 2003, 6: 54-59.
40. Pujol A, Boiso I, Benet J et al. Analysis of nine chromosome probes in first polar bodies and metaphase Ⅱ oocytes for the detection of aneuploidies. Eur J Hum Genet, 2003, 11: 325-336.
41. Cui X-S, Li X-Y, Yin X-J et al. Maternal gene transcription in mouse oocytes: genes implicated in oocyte maturation and fertilization. J Reprod Dev, 2007, 53: 405-418.
42. Gasca S, Pellestor F, Assou S et al. Identifying new human oocyte marker genes: a microarray approach. Reprod BioMed Online, 2007, 14: 175-183.
43. Nakahara K, Saito H, Saito T, et al. The incidence of apoptotic bodies: in membrane granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril, 1997, 68: 312-317.
44. Piquette GN, Tilly JL, Prichard L, et al. Detection of apoptosis in human and rat ovarian follicles. J Soc Gynecol Invest, 1994, 1: 297-301.
45. Zeuner A, Muller K, Reguszynski K, et al. Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology, 2003, 59(5-6): 1421.
46. McKenzie LJ, Pangas SA, Carson SA, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod, 2004, 19: 2869-2874.
47. Zhang X, Jafari N, Barnes RB, et al. Studies of gene expression in human cumulus cells indicate pentraxin 3 as a possible marker for oocyte quality. Fertil Steril, 2005, 83(Suppl. 1): 1169-1179.
48. Kawano Y, Narahara H, Matsui N, et al. Insulin-like growth factor-binding protein-1 in human follicular fluid： a marker for oocyte maturation．Gynecol Obstet Invest．1997， 44： 145-148．
49．Oosterhuis GJ， Vermes I， Lambalk CB， et al．Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles．Hum Reprod， 1998， 13：285-289
50．Fried G， Remaeus K， Harlin J， et al．Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization．J Assist Reprod Genet， 2003， 20： 167-176．
51．Nicholas B．Alberio R， Fouladi-Nashta AA．et al．Relationship between low-molecular-weight insulin-like growth factor-binding proteins， caspase-3 activity， and oocyte quality．Biol Reprod， 2005， 72： 796-804．
52．Chang CL， Wang TH， Horng SG， et al．The concentration of inhibin B in follicular fluid： relation to oocyte maturation and embryo development．Hum Reprod， 2002， 17：1724-1728．
53．Wen X， Tozer AJ， Butler SA， et al．Follicular fluid levels of inhibin A， inhibin B．and activin A levels reflect changes in follicl esize but are not independent markers of the oocyte's ability to fertilize．Fertil Steril， 2006， 85： 1723-1729．
54．Ebner T，Sommergruber M， Moser M， et al．Basal level of anti-Mullerian hormone is associated with oocyte quality in stimulated cycles．Hum Reprod， 2006， 21：2022-2026．
55．Laufer N， DeCherney AH，Tarlatzis BC， et al．The association between preovulatory serum17beta-estradiol patternandconceptioninhumanmenopausal gonadotropin-human chorionic gonadotropin stimulation．Fertil Steril， 1986， 46： 73-76．
56．Meyer WR， Beyler SA， Baker ST， et al．Value of estradiol response after human chorionic gonadotropin administration in predicting in vitro fertilization success．Fertil Steril， 1999， 72： 542-545．
57．Chiasson MD， Bates GW，Robinson RD．et al．Measuring estradiol levels after human chorionic gonadotropin administration for in vitro fertilization is not clinically useful．Fertil Steril， 2007， 87： 448-450．
58．陆秀娥，周馥珍，黄荷凤．体外受精-胚胎移植周期卵泡液中甾体激素水平与卵母细胞发育的关系．浙江大学学报(医学版)，2000， 29 (5)： 199-202．
59．Andersen CY．Characteristics of human follicular fluid associated with successful conception after in vitro fertilization．J Clin Endocrinol Metab， 1993， 77： 1227-1234．
60．Xia P，Younglai EV．Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment．J Reprod Fertil， 2000， 118： 229-233．
61．Ruiz Anguas J．Carballo Mondragon E， Anta Jaen E， et al．Usefulness of luteinizing hormone levels to determine the oocyte quality．Ginecol Obstet Mex．2005， 73：28-35．
62．Tsai EM， Yang CH， Chen SC．et al．Leptin affects pregnancy outcome of in vitro fertilisation and steroidogenesis of human granulosa cells．J Assist Reprod Genet， 2002，19： 169-176．
63．邵明君，叶碧绿，赵军招，等．瘦素与多囊卵巢综合征发病及体外受精结局的相关研究．生殖医学杂志，2006， 15 (5)： 293-296．
64．Barroso G， Barrionuevo M， Rao P， et al ．Vascular endothelial growth factor， nitric oxide and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients．Fertil Steril，，．1999， 72(6)： 1024-1026．
65．Wunder DM Kretschmer R， Bersinger NA．Concentrations of leptin and，C-reactive protein in serum and follicular fluid during assisted reproductive cycles．Hum Reprod，2005，20：1266-1271．
66．Seino T， Saito H， Kaneko T， et al．Eight-hydroxy-2_-deoxyguanosine in granulose cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program．Fertil Steril， 2002， 77： 1184-1190．
67．Bedaiwy MA， Falcone T， Mohamed MS， et al．Differential growth of human embryos in vitro： role of reactive oxygen species．Fertil Steril， 2004， 82： 593-600．
68．Pasqualotto EB．Agarwal A， Sharma RK， et al．Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures．Fertil Steril， 2004， 81：973-976．
69．Seli-E， Sakkas D， Behr B， et al．Noninvasive metabolomic profiling of human embryo culture media correlates with pregnancy outcome．Initial results of the metabolomics study group for ART．In： Proceedings of the 62nd annual meeting of American Society for Reproductive Medicine， 2006．
70. Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod, 2004, 19: 2319-2324.
71. Haggarty P, Wood M, Ferguson E, et al. Fatty acid metabolism in human preimplantation embryos. Hum Reprod, 2006, 21: 766-773.
72. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod, 1996, 11: 1975-1978.
73. Thomas N, Goodacre R, Timmins EM, et al. Fourier transform infrared spectroscopy of follicular fluids from large and small antral follicles. Hum Reprod, 2000, 15: 1667-1671.
74. Patrizio P, Fragouli E, Bianchi V, et al. Molecular methods for selection of the ideal oocyte. Reprod Biomed Online, 2007, 15(3): 346-353.
2．Park JY， Su YQ， Ariga M， et al．EGF-like growth factors as midiators of LH action in the ovulatory follicle．Science， 2004，303 (5658)：682-684．
3．Kawamura K， Kumagai J， Sudo S et al．Paracrine regulation of mammalian oocyte maturation and male germ cell survival．Proc Natl Acad Sci U S A， 2004．101(19)：7323-7328．
4．Ofeda SR， Romero C， Tapia C， et al．Neurotrophic and cell-cell dependent control of early follicular development．Mol cell Endocrinol， 2001，63：67-71．
5．Harel S， Jin S．Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod， 2006， 12(6)：357-365．
6．Paredes A， Romero C，Dissen GA， et al．TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary．Dev Biol， 2004， 267： 430-449．
7．Seifer DB， Feng B， Shelden RM．et al．Brain-derived neurotrophic factor： a novel human ovarian follicular protein．J Clin Endocrinol Metab， 2002， 87(2)： 655-659．
8．Seifer DB， Feng B， Shelden RM．Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol．2006， 194(4)： 1129-1134．
9．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
10．Kawamura K．Kawamura N， Mulders SM， et al．Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos．Proc Natl Acad Sci U S A， 2005， 102(26)：9206-9211．
11．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development．Reproduction， 2005，129 (4)：423-434
12．Lee E， Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
13．Sirard MA， Richard F， Blondin P， et al．Contribution of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
14．Rose CR， Blum R， Kafitz KW， et al．From modulator to mediator： rapid effects of BDNF on ion channels．Bio Essays， 2004， 26：1185-1194．
15．Fan HY， Sun QY Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals．Biol reprod， 2004， 70： 535-547．
16．Andersen CB， Sakaue H， Nedachi T，et al．Protein kinase B/Akt is essential for the insulin-but not progesterone-stimulated resumption of meiosis in Xenopus oocytes．Biochem J， 2003， 369(Pt 2)：227-238．
17．Hoshino Y， Sato E．Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes．Dev Biol， 2008， 314： 215-223．
18．Tosti E．Calcium ion currents mediating oocyte maturation events．Reprod Biol Endocrinol， 2006，4：26-37．
1．Jensen T， Johnson AL．Expression and function of brain-derived neurotrophin factor and its receptor， TrkB．in ovarian follicles from the domestic hen (Gallus gallus domesticus)．J Exp Biol， 2001，204(Pt 12)： 2087-2095．
2．Kawamura K， Kawamura N．Mulders SM， et al．Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos．Proc Natl Acad Sci U S A．2005， 102(26)： 9206-9211．
3．Dissen GA， Hirshfield AN， Malamed S， et al．Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated： changes at the time of folliculogenesis．Endocrinology， 1995， 136(10)： 4681-4692．
4．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotesbovineoocytecytoplasmiccompetenceforembryodevelopment．Reproduction， 2005， 129(4)： 423-434．
5．Lee E．Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
6．Seifer DB， Feng B， Shelden RM．Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol， 2006．194(4)： 1129-1134．
7．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
8．Harel S， Jin S， Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod， 2006， 12(6)： 357-365．
9．Seifer DB， Feng B， Shelden RM， et al．Brain-derived neurotrophic factor： a novel human ovarian follicular protein．J Clin Endocrinol Metab， 2002， 87(2)： 655-659．
10．Paredes A， Romero C， Dissen GA， et al．TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary．Dev Biol， 2004， 267 (2)：430-449．
11．Ofeda SR， Romero C， Tapia C， et al．Neurotrophic and cell-cell dependent control of early follicular development．Mol cell Endoorinol， 2000， 163(1-2)：67-71．
12．Goud PT， Goud AP， Qian C，．et al．In-vitro maturation of human germinal vesicle stage oocytes： role of cumulus cells and epidermal growth factor in the culture medium．Hum．Reprod， 1998， 13(6)： 1638-1644．
13．Demeestere Ⅰ， Gervy C， Centner J．et al．Effect of insulin-like growth factor-Ⅰ during preantral follicular culture on steroidogenesis， in vitro oocyte maturation， and embryo development in mice．Biol Reprod， 2004， 70(6)： 1664-1669．
14．Stock AE， Woodruff TK， Smith LC．Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone-and serum-free medium．Biol Reprod， 1997，56： 1559-1564．
15．Ikeda S．Ichihara-Tanaka K， Azuma T．et al．Effects of midkine during in vitro maturation of bovine oocytes on subsequent developmental competence．Biol Reprod，2000， 63：1067-1074．
1．Sirard MA， Richard F， Blondin P， et al．Contribution．，of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
2．Liu H， Krey LC， Zhang J， et al．Ooplasmic influence on nuclear function during the metaphase Ⅱ-interphase transition in mouse oocytes．Biol Reprod， 2001，65：1794-1799．
3．Salamone DF， Damiani P， Fissore RA， et al．Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised．Biol Reprod，2001，64： 1761-1768．
4．Hu Y．Betzendahl I， Cortvrindt R， et al．Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture．Hum Reprod， 2001，16：737-748．
5．Eppig JJ， O'Brien MJ．Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo．Theriogenology， 1998， 49：415-422．
6．Mermillod P， Oussaid B， Cognie Y Aspects of follicular and oocyte maturation that affect the developmental potential of embryos．J Reprod Fertil Suppl， 1999， 54：449-460．
7．Sanfins A．Lee GY， Plancha CE， et al．Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes．Biol Reprod．2003，69： 2059-2067．
8．Liu XY， Mal SF， Miao DQ， et al．Cortical granules behave differently in mouse oocytes matured under different conditions．Hum reprod．2005， 20： 3402-3413．
9．Ferreira EM， Vireque AA， Adona PR， et al．Cytoplasmic maturation of bovine oocytes：Structural and biochemical modifications and acquisition of developmental competence．Theriogenology， 2009．71 (5)： 836-848．
10．Wang Q， Sun QYEvaluation of oocyte quality： morphological， cellular and molecular predictors．Reprod Fertil Dev， 2007， 19(1)： 1-12．
11．Sun QY， Wu GM， Lai L， et al．Translocation of active mitochondria during pig oocyte maturation， fertilization and early embryo development in vitro．Reproduction， 2001， 122： 155-163．
12．Combelles CM， Racowsky C．Assessment and optimization of oocyte quality during assisted reproductive technology treatment．Semin Reprod Med， 2005， 23：277-284．
13．Sanfins A， Plancha CE， Overstrom EW， et al．Meiotic spindle morphogenesis in in vivo and in vitro matured mouse oocytes： insights into the relationship between nuclear and cytoplasmic quality．Hum Reprod， 2004， 19(12)： 2889-2899．
14．Gilchrist RB， Thompson JG．Oocyte maturation： Emerging concepts and technologies to improve developmental potential in vitro．Theriogenology， 2007， 67： 6-15．
15．张玲，朱桂金．丁内酯-Ⅰ对小鼠卵母细胞体外成熟及发育能力的影响．生殖与避孕，2005， 25 (7)： 396-399．
16．Combelles CM， Albertini DF．Microtubule patterning during meiotic maturation in mouse oocytes is determined by cell cycle-specific sorting and redistribution of gamma-tubulin．Dev Biol．2001，239： 281-294．
17．Patrizio P， Fragouli E， Bianchi V，et al．Molecular methods for selection of the ideal oocyte．Reprod Biomed Online， 2007， 15(3)： 346-353．
18．Leader B， Lim H， Carabatsos MJ， et al．Formin-2， polyploidy．hypofertility and positioning of the meiotic spindle in mouse oocytes．Nat Cell Biol， 2002， 4： 921-928．
19．Sun QY， Schatten H．Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction, 2006. 13: 193-205.
20. Tapia-Arancibia L, Rage F, Givalois L, et al. Physiology of BDNF: focus on hypothalamic function. Front Neuroendocrinol, 2004, 25(2): 77-107.
21. Rose CR, Blum R, Kafitz Kw, Kovalchuk Y, Konnerth A. From modulator to mediator: rapid effects of BDNF on ion channels. BioEssays, 2004, 26: 1185-1194.
22. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol reprod, 2004, 70: 535-547.
23. Andersen CB, Sakaue H, Nedachi T, et al. Protein kinase B/Akt is essential for the insulin-but not progestone-stimulated resumption of meiosis in Xenopus oocytes. BioChem J, 2003, 369: 227-238.
24. Hoshino Y, Sato E. Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes. Dev Biol, 2008, 314: 215-223.
25. Han SJ, Vaccari S, Nedachi T, et al. Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation. EMBO J, 2006, 25(24): 5716-5725.
26. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005, 281: 160-170.
27. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
28. Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179:25-34.
1. Friedman WJ, Greene LA. Neurotrophin Signaling via Trks and p75. Exp Cell Res, 1999,253: 131-142.
2. Tapia-Arancibia L, Rage F, Givalois L, et al. Physiology of BDNF: focus on hypothalamic function. Front Neuroendocrinol, 2004, 25(2): 77-107.
3. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biol reprod 2004; 70: 535-547.
4. Su YQ, Wigglesworth K, Pendola FL, et al. Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology, 2002, 143: 2221-2232.
5. Hoshino Y, Sato E. Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes. Dev Biol, 2008, 314: 215-223.
6. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005/281: 160-170.
7. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
8. Shimada M, Ito J, Yamashita Y, et al. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179: 25-34.
9. Andersen CB, Sakaue H, Nedachi T, et al. Protein kinase B/Akt is essential for the insulin-but not progestone-stimulated resumption of meiosis in Xenopus oocytes. BioChem J, 2003, 369: 227-238.
10. Tosti E. Calcium ion currents mediating oocyte maturation events. Reprod Biol Endocrinol, 2006, 4: 26-37.
11. Ofeda SR, Romero C, Tapia C, et al. Neurotrophic and cell-cell dependent control of early follicular development. Mol cell Endoorinol, 2000, 163 (1-2) : 67-71.
12. Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J, 1999, 18(3): 616-622.
13. Kawamura K, Kawamura N, Mulders SM, et al. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci U S A, 2005, 102(26): 9206-9211.
14. Lengyel F, Vertes Z, Kovacs KA,et al. Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus. J. Steroid. Biochem. Mol Biol, 2004, 91, 285-288.
15. Viniegra JG, Martinez N, Modirassari P, et al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem, 2005, 280: 4029-4036.
16. Kalous J, Solc P, Baran(?) V, et al. PKB/AKT is involved in resumption of meiosis in mouse oocytes. Biol cell, 2006, 98, 111-123.
17. Patrizio P, Fragouli E, Bianchi V, et al. Molecular methods for selection of the ideal oocyte. Reprod Biomed Online, 2007, 15(3): 346-353.
18. Su YQ, Wigglesworth K, Pendola FL, et al. Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology, 2002, 143: 2221-2232.
19. Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem, 2003, 72: 609-642.
1．Martins da Silva Sj， Gardner JO， Taylor JE， et al．Brain-derived neurotrophic factor promotesbovineoocytecytoplasmiccompetenceforembryodevelopment．Reproduction， 2005， 129(4)： 423-434．
2．Tapia-Arancibia L， Rage F， Givalois L．et al．Physiology of BDNF： focus on hypothalamic function．Front Neuroendocrinol，2004， 25(2)： 77-107．
3．付娟，邢福棋．脑源性神经营养因子与卵巢．国外医学计划生育分册，2003， 22(4)： 207-210．
4．Lee R， Kermani P．Teng KK， et al．Regulation of cell survival by secreted proneurotrophins．Science， 2001，294： 1945-1948．
5. Ofeda SR, Romero C, Tapia C, et al. Neurotrophic and cell-cell dependent control of early follicular development. Mol cell Endoorinol, 2000, 163 (1-2) : 67-71.
6. Bibel M, Hoppe E, Barde YA. Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J, 1999, 18(3): 616-622.
7. Mischel PS, Smith SG, Vining ER,et al. The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA. J Biol Chem, 2001, 276(14): 11294-11301.
8. Eide FF, Vining ER, Eide BL,et al. Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J Neurosci, 1996, 16 (10): 3123-3129.
9. Paredes A, Romero C, Dissen GA, et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary. Dev Biol, 2004, 267 (2) : 430-449
10. Kerr B, Garcia-Rudaz C, Dorfman M, et al. TrkA and TrkB receptors facilitate follicle assembly and early follicular development in the mouse ovary. Reproduction, 2009. [Epub ahead of print]
11. Dissen GA, Hirshfield AN, Malamed S, et al. Expression of:heurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis. Endocrinology, 1995, 136(10): 4681-4692.
12. Dissen GA, Garcia-Rudaz C, Ojeda SR. Role of neurotrophic factors in early ovarian development. Semin Reprod Med, 2009, 27(1): 24-31.
13. Kawamura K, Kawamura N, Mulders SM, et al. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci U S A, 2005, 102(26): 9206-9211.
14. Seifer DB, Feng B, Shelden RM, et al. Brain-derived neurotrophic factor: a novel human ovarian follicular protein. J Clin Endocrinol Metab, 2002, 87(2): 655-659.
15. Seifer DB, Lambert-Messerlian G, Schneyer AL. Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women. Fertil Steril, 2003, 79: 451-452.
16. Seifer DB, Feng B, Shelden RM. Immunocytochemical evidence for the presence and location of the neurotrophin-Trk receptor family in adult human preovulatory ovarian follicles．Am J Obstet Gynecol， 2006．194(4)： 1129-1134．
17．付娟，邢福祺，陈士岭．酪氨酸蛋白激酶受体B在人卵泡中的表达及意义．第一军医大学学报，2003， 23 (2)：162-164．
18．Harel S， Jin S， Fisch B， et al．Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults．Mol Hum Reprod．2006， 12(6)：357-365．
19．Lee E， Jeong Yi， Park SM， et al．Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes．Reproduction， 2007， 134(3)： 405-414．
20．Jensen T， Johnson AL．Expression and function of brain-derived neurotrophin factor and its receptor， TrkB， in ovarian follicles from the domestic hen (Gallus gallus domesticus)．J Exp Biol， 2001，204(Pt 12)： 2087-2095
21．付娟，邢福祺，陈士岭等．促性腺激素对人卵巢脑源性神经营养因子及其受体表达的影响．生殖与避孕，2003，23 (3)：149-152．
22．Begliuomini S， Casarosa E， Pluchino N．et al．Influence of endogenous and exogenous sex hormones on plasma brain-derived neurotrophic factor．Hum Reprod， 2007， 22(4)：995-1002．
23．Monteleone P， Artini PG，SimiG，et al．Brain derived neurotrophic factor circulating levels in patients undergoing IVF．J Assist Reprod Genet， 2007， 24： 477-480．
24．Piccinni A， Marazziti D， Del Debbio A， et al．Diurnal variation of plasma brain-derived neurotrophic factor (BDNF) in humans： an analysis of sex differences．Chronobiol Int，2008．25(5)： 819-826．
25．Huang EJ．Reichardt LF．Trk receptors： roles in neuronal signal transduction．Annu Rev Biochem．2003， 72： 609-642．
26．Fan HY， Sun QY．Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals．Biol reprod， 2004， 70： 535-547．
27．Su YQ， Wigglesworth K， Pendola FL， et al．Mitogen-activated protein kinase activaty in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse．Endocrinology， 2002， 143：2221-2232．
28．Hoshino Y， Sato E．Protein kinase B (PKB/Akt) is required for the completion of meiosis in mouse oocytes．Dev Biol， 2008， 314： 215-223．
29. Reddy P, Shen L, Ren C, et al. Activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Dev Biol, 2005, 281: 160-170.
30. Tomek W, Smiljakovic T. Activation of Akt (protein kinase B) stimulates metaphase Ⅰ to metaphase Ⅱ transition in bovine oocytes. Reproduction, 2005, 130: 423-430.
31. Shimada M, Ito J, Yamashita Y, et al. Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol, 2003, 179: 25-34.
32. Tosti E. Calcium ion currents mediating oocyte maturation events. Reprod Biol Endocrinol, 2006, 4: 26-37.
33. Park C, Choi WS, Kwon H, et al. Temporal and spatial expression of neurotrophins and their receptors during male germ cell development. Mol Cells, 2001, 12(3):360-367.
34. Wheeler EF, Bothwell M. Spatiotemporal patterns of expression of NGF and the low-affinity NGF receptor in rat embryos suggest functional, roles in tissue morphogenesis and myogenesis. JNeurosci, 1992, 12(3): 930-945.
35. Muller D, Davidoff MS, Bargheer O, et al. The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells. Histochem Cell Biol, 2006, 126(2): 199-211.
1．Sirard MA．Richard F， Blondin P， et al．Contribution of the oocyte to embryo quality．Theriogenology， 2006， 65(1)： 126-136．
2．Ferreira EM， Vireque AA， Adona PR， et al．Cytoplasmic maturation of bovine oocytes： Structural and biochemical modifications and acquisition of developmental competence．Theriogenology， 2009．71 (5)： 836-848．
3．Wang Q， Sun QYEvaluation of oocyte quality： morphological， cellular and molecular predictors．Reprod Fertil Dev， 2007， 19(1)： 1-12．
4．Krisher R L．The effect of oocyte quality on development．J Anim Sci， 2004，82： E14-E23.
5. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryoquality. Hum Reprod, 1997, 12(8): 1750-1755.
6. Ten J, Mendiola J, Vioque J, et al. Donor oocyte dysmorphisms and their influence on fertilization and embryo quality. Reprod Biomed Online, 2007, 14(1): 40-48.
7. Kahraman S, Yakin K, Donmez E, et al. Relationship between granular cytoplasm of oocytes and pregnancy outcome following intracytoplasmic sperm injection. Hum. Reprod, 2000,15:2390-2393.
8. Balaban B, Urman B. Effect of oocyte morphology on embryo development and implantation.Reprod Biomed Online, 2006, 12(5):608-615.
9. Gabrielsen, A, Lindenberg, S, Petersen, K. The impact of the zona pellucida thickness variation of human embryos on pregnancy outcome in relation to suboptimal embryo development. A prospective randomized controlled study. Hum Reprod, 2001, 16: 2166-2170.
10. Hu Y, Betzendahl I, Cortvrindt R, et al. Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture. Hum Reprod , 2001, 16: 737-748.
11. Mermillod P, Oussaid B, Cognie Y. Aspects of follicular and oocyte maturation that affect the developmental potential of embryos. J Reprod Fertil Suppl, 1999, 54: 449-460.
12. Cooke S, Tyler J P, Driscoll G L. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod, 2003, 18: 2397-2405.
13. Sanfins A, Lee GY, Plancha CE, et al. Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes. Biol Reprod, 2003, 69: 2059-2067.
14. Rossi G, Macchiarelli G, Palmerini MG, et al. Meiotic spindle configuration is differentially influenced by FSH and epidermal growth factor during in vitro maturation of mouse oocytes. Hum Reprod, 2006, 21: 1765-1770.
15. Moon J H, Hyun C S, Lee S W, et al. Visualization of the metaphase Ⅱ meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum. Reprod, 2003, 18: 817-820.
16. Wang WH, Meng L, Hackett R J, et al. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod, 2001,16: 1464-1468.
17. De Santis L, Cino I, Rabellotti E, et al. Polar body morphology and spindle imaging as predictors of oocyte quality. Reprod Biomed Online, 2005, 11(1): 36-42.
18. Lonergan P, Monaghan P, Rizos D, et al. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mo. Reprod Dev, 1994, 37: 48-53.
19. Borini A, Maccolini A, Tallarini A, et al. PerifoUicular vaseularity and its relationship with oceyte maturity and IVF outcome. Ann N Y Acad Sci, 2001, 943: 64-67.
20. Wilding M, Dale B, Marino M, et al. Mitochondrial aggregation.patterns and activity in human oocytes and preimplantation embryos. Hum Reprod, 2001,16: 909-917.
21. Singh R, Sinclair KD. Metabolomics: approaches to assessing:* iooeyte and embryo quality.Theriogenology, 2007, 68 Suppl 1: S56-62.
22. Bavister BD, Squirrell JM. Mitochondrial distribution and function in oocytes and early embryos. Hum Reprod, 2000, 15: 189-198.
23. Ludwig M, Schopper B, AI-Hasani S, et al. Clinical use of a pronuclear stage score following intracytoplasmic sperm injection: impact on pregnancy rates under the condition of the German embryo protection law. Hum Reprod, 2000, 15(2): 325-329.
24. Brevini T A, Vassena R, Francisci C, et al. Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol Reprod, 2005, 72: 1218-1223.
25. Keefe D, Niven-Fairchild T, Powel S, et al .Mitochondrial deoxyribonucleic acid deletions in oocytes of reproductively aging women. Fertil Steril, 1995, 64(3): 577-583.
26. HsiehRH, Au HK, Yeh TS, et al. Decreased expression of mitochondrial genes in human unfertilized oocytes and arrested embryos. Fertil Steril, 2004, 81 [Suppl 1]: 912-918.
27. Van Blerkom J. Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction, 2004, 128(3): 269-280.
28. Santos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril, 2006, 85: 584-591.
29. Stojkovic M, Machado SA, Stojkovic P, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod, 2001, 64: 904-909.
30. Tamassia M, Nuttinck F, May-Panloup P, et al. In vitro embryo production efficiency in cattle and its association with oocyte adenosine triphosphate content, quantity of mitochondrial DNA, and mitochondrial DNA haplogroup. Biol Reprod, 2004, 71: 697-704.
31. Liu XY, Mal SF, Miao DQ, et al. Cortical granules behave differently in mouse oocytes matured under different conditions. Hum reprod, 2005, 20: 3402-3413.
32. Ferrandi B, Cremonesti G, Geiger R, et al. Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation. Acta Histochem, 1993, 95: 89-96.
33. Ericsson SA, Boice ML, Funahashi H, et al. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology, 1993, 39: 214.
34. Alm H, Torner H, Lohrke B, et al. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresyl blue staining before IVM as indicator for glucose-6- phosphate dehydrogenase activity. Theriogenology, 2005, 63: 2194-2205.
35. El Shourbagy SH, Spikings EC, Freitas M, et al. Mitochondria directly influence fertilization outcome in the pig. Reproduction, 2006, 131: 233-245.
36. Baart EB, van den Berg I, Martini E, et al. FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos: the added value of extended aneuploidy detection. Prenatal Diagnosi, 2007, 27: 55-63.
37. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod, 2000, 6: 1055-1062.
38. Munne S, Chen S, Fischer J et al. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertil Steril, 2005, 84: 331-335.
39. Kuliev A, Cieslak J, Ilkevitch Y, et al. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod BioMed Online, 2003, 6: 54-59.
40. Pujol A, Boiso I, Benet J et al. Analysis of nine chromosome probes in first polar bodies and metaphase Ⅱ oocytes for the detection of aneuploidies. Eur J Hum Genet, 2003, 11: 325-336.
41. Cui X-S, Li X-Y, Yin X-J et al. Maternal gene transcription in mouse oocytes: genes implicated in oocyte maturation and fertilization. J Reprod Dev, 2007, 53: 405-418.
42. Gasca S, Pellestor F, Assou S et al. Identifying new human oocyte marker genes: a microarray approach. Reprod BioMed Online, 2007, 14: 175-183.
43. Nakahara K, Saito H, Saito T, et al. The incidence of apoptotic bodies: in membrane granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril, 1997, 68: 312-317.
44. Piquette GN, Tilly JL, Prichard L, et al. Detection of apoptosis in human and rat ovarian follicles. J Soc Gynecol Invest, 1994, 1: 297-301.
45. Zeuner A, Muller K, Reguszynski K, et al. Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology, 2003, 59(5-6): 1421.
46. McKenzie LJ, Pangas SA, Carson SA, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod, 2004, 19: 2869-2874.
47. Zhang X, Jafari N, Barnes RB, et al. Studies of gene expression in human cumulus cells indicate pentraxin 3 as a possible marker for oocyte quality. Fertil Steril, 2005, 83(Suppl. 1): 1169-1179.
48. Kawano Y, Narahara H, Matsui N, et al. Insulin-like growth factor-binding protein-1 in human follicular fluid： a marker for oocyte maturation．Gynecol Obstet Invest．1997， 44： 145-148．
49．Oosterhuis GJ， Vermes I， Lambalk CB， et al．Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles．Hum Reprod， 1998， 13：285-289
50．Fried G， Remaeus K， Harlin J， et al．Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization．J Assist Reprod Genet， 2003， 20： 167-176．
51．Nicholas B．Alberio R， Fouladi-Nashta AA．et al．Relationship between low-molecular-weight insulin-like growth factor-binding proteins， caspase-3 activity， and oocyte quality．Biol Reprod， 2005， 72： 796-804．
52．Chang CL， Wang TH， Horng SG， et al．The concentration of inhibin B in follicular fluid： relation to oocyte maturation and embryo development．Hum Reprod， 2002， 17：1724-1728．
53．Wen X， Tozer AJ， Butler SA， et al．Follicular fluid levels of inhibin A， inhibin B．and activin A levels reflect changes in follicl esize but are not independent markers of the oocyte's ability to fertilize．Fertil Steril， 2006， 85： 1723-1729．
54．Ebner T，Sommergruber M， Moser M， et al．Basal level of anti-Mullerian hormone is associated with oocyte quality in stimulated cycles．Hum Reprod， 2006， 21：2022-2026．
55．Laufer N， DeCherney AH，Tarlatzis BC， et al．The association between preovulatory serum17beta-estradiol patternandconceptioninhumanmenopausal gonadotropin-human chorionic gonadotropin stimulation．Fertil Steril， 1986， 46： 73-76．
56．Meyer WR， Beyler SA， Baker ST， et al．Value of estradiol response after human chorionic gonadotropin administration in predicting in vitro fertilization success．Fertil Steril， 1999， 72： 542-545．
57．Chiasson MD， Bates GW，Robinson RD．et al．Measuring estradiol levels after human chorionic gonadotropin administration for in vitro fertilization is not clinically useful．Fertil Steril， 2007， 87： 448-450．
58．陆秀娥，周馥珍，黄荷凤．体外受精-胚胎移植周期卵泡液中甾体激素水平与卵母细胞发育的关系．浙江大学学报(医学版)，2000， 29 (5)： 199-202．
59．Andersen CY．Characteristics of human follicular fluid associated with successful conception after in vitro fertilization．J Clin Endocrinol Metab， 1993， 77： 1227-1234．
60．Xia P，Younglai EV．Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment．J Reprod Fertil， 2000， 118： 229-233．
61．Ruiz Anguas J．Carballo Mondragon E， Anta Jaen E， et al．Usefulness of luteinizing hormone levels to determine the oocyte quality．Ginecol Obstet Mex．2005， 73：28-35．
62．Tsai EM， Yang CH， Chen SC．et al．Leptin affects pregnancy outcome of in vitro fertilisation and steroidogenesis of human granulosa cells．J Assist Reprod Genet， 2002，19： 169-176．
63．邵明君，叶碧绿，赵军招，等．瘦素与多囊卵巢综合征发病及体外受精结局的相关研究．生殖医学杂志，2006， 15 (5)： 293-296．
64．Barroso G， Barrionuevo M， Rao P， et al ．Vascular endothelial growth factor， nitric oxide and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients．Fertil Steril，，．1999， 72(6)： 1024-1026．
65．Wunder DM Kretschmer R， Bersinger NA．Concentrations of leptin and，C-reactive protein in serum and follicular fluid during assisted reproductive cycles．Hum Reprod，2005，20：1266-1271．
66．Seino T， Saito H， Kaneko T， et al．Eight-hydroxy-2_-deoxyguanosine in granulose cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program．Fertil Steril， 2002， 77： 1184-1190．
67．Bedaiwy MA， Falcone T， Mohamed MS， et al．Differential growth of human embryos in vitro： role of reactive oxygen species．Fertil Steril， 2004， 82： 593-600．
68．Pasqualotto EB．Agarwal A， Sharma RK， et al．Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures．Fertil Steril， 2004， 81：973-976．
69．Seli-E， Sakkas D， Behr B， et al．Noninvasive metabolomic profiling of human embryo culture media correlates with pregnancy outcome．Initial results of the metabolomics study group for ART．In： Proceedings of the 62nd annual meeting of American Society for Reproductive Medicine， 2006．
70. Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod, 2004, 19: 2319-2324.
71. Haggarty P, Wood M, Ferguson E, et al. Fatty acid metabolism in human preimplantation embryos. Hum Reprod, 2006, 21: 766-773.
72. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod, 1996, 11: 1975-1978.
73. Thomas N, Goodacre R, Timmins EM, et al. Fourier transform infrared spectroscopy of follicular fluids from large and small antral follicles. Hum Reprod, 2000, 15: 1667-1671.
74. Patrizio P, Fragouli E, Bianchi V, et al. Molecular methods for selection of the ideal oocyte. Reprod Biomed Online, 2007, 15(3): 346-353.