人卵泡发育超微结构、基因表达谱的研究及未成熟卵体外培养的临床应用
|
摘要
研究背景
对卵母细胞和卵泡发育、成熟机制的研究是人类揭示自身繁衍的奥秘、提高人口素质的需要。自从20世纪30年代以来，随着物理化学技术，尤其是电子显微镜技术的发明和分子生物学技术的不断发展，打破了光学显微研究细胞的局限而进入了超微结构和分子生物水平的领域，为包括生殖细胞的来源、原始生殖细胞分化、始基卵泡由静止池进入生长发育阶段的动因、初级卵泡发育为次级卵泡和成熟卵泡过程的形态学变化规律和内分泌环境、白分泌和旁分泌作用等研究提供了技术平台。尤其是1978年世界首例体外受精—胚胎移植(in vitro fertilization-embryo transfer，ⅣF-ET)婴儿的诞生，一方面为人类生殖机制的深入研究提供了契机，另一方面更激起了生殖学者对人类生殖机制了解的迫切需求，同期遗传分子生物学技术的迅猛发展、生物芯片技术的出现，更为我们探索生命发生、进化、生长、发育、凋亡的规律提供了有力的技术支撑。
目前ⅣF周期的胚胎着床率大约在20％左右，妊娠率在30％～40％，活胎出生率在25％左右，主要原因之一在于促排卵周期所获卵子有相当一部分发育异常，不能受精或不能形成高质量的胚胎。1983年取得的卵母细胞体外成熟(ⅣM)的成功，由于不需要促排卵，简化了ⅣF程序、节约了治疗费用、避免了ⅣF周期严重卵巢过度刺激综合症的发生，因而受到了人们的关注。然而，20多年来，ⅣM技术的应用一直有限，据2005年统计仅出生子代300名，原因在于ⅣM技术至今未克服卵母细胞体外成熟过程中胞核和胞浆成熟不一致的难题，对卵母细胞发育、受精、胚胎发育起关键作用的胞浆成熟远远落后于胞核。对卵母细胞／卵泡发育、成熟机制和胚胎发育机制的研究，将有助于提高辅助生育技术(ART)的成功率和子代健康状况。从长远来说有助于我们对人类生殖繁衍奥秘的了解，为妇女生殖健康措施制定提供理论指导。
卵母细胞在卵巢中进行减数分裂的过程称为卵的发生，这个过程包括增殖期、生长期以及成熟期3个阶段。卵泡的发生、发育和成熟包括卵泡的募集、选择和优势化。卵母细胞的成熟与其周围的卵泡细胞的作用密不可分，卵母细胞发育伴随了卵泡的增大，当卵泡发育到一定大小时才发生卵母细胞核／质的成熟。卵泡发育根据形态和功能可分为以下3个阶段：(1)静止卵泡：包括原始卵泡、中间卵泡和初级卵泡；(2)早期生长卵泡：包括次级卵泡、窦前卵泡、早期窦卵泡、选择卵泡；(3)窦卵泡：直径达2mm，对FSH敏感性增加，依赖FSH继续发育，继后受LH峰作用最终成熟。
静止卵泡是非激素依赖性，受遗传因素和局部调节因子的影响，进行分化、增殖。从始基卵泡发育到次级卵泡机制不清，1991年Horie等证实由颗粒细胞产生的Kit Ligand(KL)作用于卵泡膜间质细胞的相应受体(c-Kit受体)，在无Gn的情况下单独刺激静止卵泡进入生长发育期，窦前卵泡的卵母细胞直径100～130μm，已达成熟期大小，直径2mm的窦卵泡颗粒细胞FSH受体充足，已具备了体外成熟的条件。从2mm到18mm需要25天时间，后15天相当于月经周期的卵泡期。窦卵泡生长至直径10-14mm时卵母细胞重新启动减数分裂，但仍停留在生殖泡期，发育至排卵前卵泡，直径约为15-25mm时受促黄体生成素(LH)作用，卵泡发生生殖泡破裂(GVBD)，并成为成熟卵(MⅡ)，发生排卵。卵泡进入生长状态(次级卵泡2层GC出现)至发育成熟共需85天时间。
处于MⅡ期的卵母细胞光镜下直径约150μm，从外到内，由透明带(ZP)、卵周间隙、卵黄膜、卵浆(亦称卵母细胞皮质)、细胞核及核仁组成。卵黄膜与透明带之间为充满液体的卵周间隙，内见圆形第一极体。胞浆匀称，浅灰色，颗粒均匀。当卵母细胞形态异常、胞质分布异常以及第一极体形态发生改变时将导致受精能力和进一步胚胎发育能力的下降，但这种改变伴随着的超微结构改变尚不清楚。
卵母细胞的大多数细胞器最初分布于近核区，随着卵母细胞生长，线粒体、高尔基体、粗面内质网、皮质颗粒等细胞器不断丰富和增多，并逐渐向皮质区迁移，在细胞中央形成不含细胞器的“透明区”，卵母细胞成熟后，皮质颗粒排列在质膜下，皮质颗粒的大量增加和迁移并沿质膜下呈线性排列是卵母细胞胞质成熟的一个重要标志。从初级卵母细胞到MⅡ期卵母细胞，其胞膜表面微绒毛的数量不断增多，微绒毛长度逐渐增加，至完全成熟后微绒毛数量下降。
在卵泡发育过程中卵泡细胞线粒体和粗面内质网丰富，颗粒细胞和卵母细胞伸出突起穿过透明带相互联系。卵母细胞、卵丘细胞和卵泡细胞之间存在广泛的缝隙连接形成一个完整的功能联合体，这种结构一直至卵母细胞恢复减数分裂前才发生松解。
在卵泡／卵母细胞的发育过程中卵巢局部的微环境参与了整个过程，有研究发现卵泡颗粒细胞产生的抑制素A、抑制素B和激活素A是卵母细胞成熟基本细胞因子，胰岛素样生长因子Ⅰ／Ⅱ、表皮生长因子、转化生长因子、骨形成蛋白、生长与分化因了、肿瘤坏死因子、白介素、抗苗勒管激素、卵细胞成熟抑制因子、有丝分裂原激活蛋白激酶、促成熟因子、连接蛋白43、生长与分化激素、妊娠相关血浆蛋白a、血管上皮生长因子、磷脂酰肌醇3激酶(PI3K)等对卵母细胞／卵泡发育的启动、生长、成熟起着极为重要的作用，当上述物质缺失或编码基因突变将导致卵细胞或卵泡发育、成熟障碍。
虽然已明确了卵泡发育的基本形态学改变和FSH、LH、T、E_2等激素的周期性变化，并对影响卵母细胞成熟的卵巢内微环境因素的研究取得了一定的进展，尝试在卵母细胞体外成熟培养中添加上述的某些物质，但仍未达到生理状态的成熟。卵泡／卵母细胞发生、发育、成熟过程极为复杂，包含其中的分子可能有数十种、数百种、甚至数千种?由于卵泡壁层颗粒细胞、膜细胞、卵丘颗粒细胞和卵母细胞之间众多生长因子、细胞因子的相互调节作用机制仍未完全清楚，对于ART及其衍生技术的不断涌现，卵泡和卵母细胞受超生理剂量的促性激素和性激素的影响以及在体外培养过程中的一系列超微结构的形态学变化更缺乏了解。因此，我们试图从未成熟卵泡和成熟卵泡颗粒细胞基因表达差异、卵细胞超微结构形态的改变研究卵母细胞、卵泡生长、发育、成熟机制，分析参与卵母细胞／卵泡成熟的相关因素，获取卵母细胞成熟的相关信息，为建立合理的ⅣM体系提供理论支持，探索未成熟卵获取周期准备方案、取卵时机和体外培养条件。
一、人未成熟卵和成熟卵卵丘复合物超微结构研究
目的
观察人卵丘复合物(oocyte-cumulus complex，OCCs)在体内和体外从未成熟发育至成熟的超微结构变化，并比较自然状态和常规促排卵ⅣF／ICSI周期未成熟OCCs超微结构的区别，分析不同条件和环境对卵细胞质量的影响。
方法：
用光镜分辨生发泡期(GⅤ期)、生发池破裂期(MⅠ期)的未成熟卵和达到MⅡ期的成熟卵。标本来自妇科腹腔镜手术的自然周期、ⅣF／ICSI周期、ⅣM周期捐赠的卵丘复合物(OCCs)。对妇科腹腔镜手术的自然周期未成熟卵(GⅤ期)、促排卵周期未成熟卵(GⅤ／MⅠ)、促排卵周期体内成熟卵(MⅡ)、体外成熟培养成熟卵(MⅡ)各3枚，分别经透射电镜技术处理，观察OCCs的超微结构。
结果
不同发育条件、发育阶段人卵丘颗粒细胞发育、卵母细胞透明带、微绒毛、皮质颗粒、线粒体结构、溶酶体、内质网、高尔基体、脂滴空泡和中心体等结构和分布方面均有差异。
1．自然周期的GⅤ期卵与常规促排卵ⅣF／ICSI周期获取的GⅤ期OCCs在超微结构方面存在显著差异。自然周期GⅤ期卵有丰富的脂滴、空泡，核仁清晰可见，无卵周间隙，透明带致密、较薄，高尔基体丰富，卵丘细胞与卵丘细胞之间以及卵丘细胞与卵母细胞之间存在广泛的缝隙连接，为一完整的功能合胞体。常规促排卵ⅣF／ICSI周期的GⅤ期卵丘细胞与卵母细胞连接松散，出现无结构的间隙，颗粒细胞染色质边聚于核膜等凋亡改变，卵母细胞皮质颗粒略有增多，微绒毛细长而竖立伸入透明带，脂滴减少，核膜皱褶出现，已出现受LH作用的改变。
2．ⅣF／ICSI周期体内未成熟卵母细胞(MⅠ期)卵丘细胞粗面内质网、线粒体、高尔基体、溶酶体、微管及大颗粒脂滴等细胞器极为丰富，偶见髓样小体；卵母细胞细胞器增多，线粒体、溶酶体、等结构逐渐移向卵母细胞皮质区，卵母细胞内皮质颗粒增多，质膜边缘略有增加；卵母细胞膜与透明带间出现卵周间隙，内见泡状结构；卵细胞胞核消失；透明带明显增厚，约为10～12μm，结构松散；微绒毛增粗，竖立、伸入透明带。
3．ⅣF／ICSI周期体内成熟卵母细胞(MⅡ期)胞质内为散在分布数目较多的细小空泡状结构，透明带逐渐增宽、松散；卵周间隙出现、增宽，以第一极体排出处最明显，微绒毛自透明带回缩，皮质颗粒丰富，向细胞边缘聚集，并呈线状排列在卵膜边缘；粗面内质网和高尔基体减少，在细胞中央出现不含细胞器的“透明区”；线粒体减少不明显、结构仍然清晰可见；而体外成熟卵母细胞线粒体数目减少，结构模糊，并出现较多泡状结构的滑面内质网，皮质颗粒较少；卵丘细胞线粒体肿胀、嵴消失，染色质发生异质化、边缘化，部分颗粒细胞核消失、细胞变型、质膜不完整等凋亡改变明显。
结论
1．经常规促排卵的IVF／ICSI周期，在大部分卵母细胞成熟的情况下产生的GV期卵丘复合物，由于受超生理剂量FSH以及hCG的影响，卵丘细胞与卵母细胞连接松散，将导致卵母细胞过早缺乏颗粒细胞通过缝隙连接输送的营养以及卵母细胞皮质颗粒过早出现，可能是受精率和胚胎发育能力下降的原因。超生理状态的激素坏境将影响发育延迟卵母细胞体外成熟的能力。因此，来自于常规促排卵周期的GV卵不适合进一步的培养，用于胚胎移植。
2．促排卵周期MI期卵丘细胞呈旺盛的增殖和功能活动状态，卵母细胞内的胞质结构、皮质颗粒、透明带等已发生了适合于卵母细胞成熟的显著的形态学改变，为卵母细胞成熟做好的充分的准备，具有进一步发育的潜能。
3．经体外成熟母细胞滑面内质网扩张，线粒体数目减少、结构模糊，脂滴减少，皮质颗粒减少，而且卵丘颗粒细胞凋亡改变明显，可能是卵母细胞质量下降、使其进一步发育能力受损的原因。
二、人类未成熟卵泡和成熟卵泡颗粒细胞基因表达差异
目的
分析人未成熟卵泡和成熟卵泡颗粒细胞基因表达差异，探讨卵泡颗粒细胞在卵泡／卵母细胞成熟过程中可能参与的分子机制。
方法
获取IVM周期未成熟卵泡(直径5～12mm)的卵泡液和常规促排卵IVF周期成熟卵(直径≥14mm)的卵泡液，分离颗粒细胞，抽提、纯化mRNA。将未成熟卵泡颗粒细胞设为研究组，成熟卵泡颗粒细胞设为对照组，采用含8300个人类基因的基因芯片进行差异性表达研究，并对其中有差异性表达的10个基因采用荧光实时定量技术(Real-time RT-PCR)进行验证。
结果
1．经基因芯片筛查，人未成熟卵泡和成熟卵泡颗粒细胞比较有462个基因表达存在差异，其中190个基因为上调表达，272个基因为下调表达。对这462个差异表达基因进行分类：原癌基因和抑癌基因15个；离子通道和运输蛋白13个；细胞周期蛋白类15个；外压反应蛋白1个；细胞骨架和运动蛋白18个；细胞凋亡相关的蛋白4个；DNA合成和修复、重组蛋白8个；DNA结合、转录和转录因子15个；细胞受体蛋白11个；免疫相关蛋白20个；细胞信号和传递蛋白54个；代谢相关调节蛋白66个；蛋白翻译合成调节因子36个；发育相关蛋白9个；其它蛋白92个，其中某些基因兼有2种或2种以上功能。尚有166个基因未分类或功能未明。
2．应用Real-time RT-PCR方法，按下调和上调表达基因的比例，分别选取其中6个下调基因和4个上调基因进行验证。6个下调基因STAR、MPHOSPH6、CALM1、MAML1、CD53和RRAGA基因芯片比率分别为0.16、0.219、0.214、0.319、0.183和0.411，Real-time RT-PCR的比率分别为0.166、0.086、0.213、0.186、0.177、0.345；4个上调基因ICAM1、CYR61、ZNF264和STMN1基因芯片的比率分别为2.385、3.619、3.992和10.245，Real-time RT-PCR的比率分别为2.28、2.023、2.30和5.07，比率趋势两者相符。10个基因的Real-time RT-PCR扩增产物经统计学分析，ICAM1表达无差异(P=0.11)，其他9个基因表达均存在显著差异(P＜0.001)。
3．排卵前未成熟卵颗粒细胞的基因表达分析，提示以调节颗粒细胞增殖、线粒体复制、细胞间缝隙连接、雌激素分泌、能量合成的基因上调表达为主。生长因子类如：CTGF、TIEG、PDGFC、FGFR-1、CGR19等；周期调节蛋白及凋亡相关因子如：SMC1L1、HMG2，PTP4A(?)、cyclin B、CDK9、STMN1、melanoma antigen等；与信息转导和细胞连接相关的基因：PRSS11、PTP4A1、CYR61、GAB1、ICAM1(CD54)、Integrins-β8、connexin 43等。与细胞骨架形成有关的微管蛋白等也有优势表达，如XPO1、GPAT、SPP1、LOC51174、MIP-T3等。与刺激雌激素合成相关的调节因子如CYP19、IRS1、ADRBK2、CRHBP等。
4．成熟卵颗粒细胞基因表达分析，提示以调节类固醇激素合成、线粒体合成的能量转运、细胞信息转导、细胞外基质形成和粘液分泌的基因上调表达为主。与信号转导相关的基因如：PTPRG、PRKCD、KIAA010、FDPS、IMPA1、TPI1、PSPHL、PIP5K2B等；细胞外基质形成、粘液分泌相关的基因：PTX3、CALM1、MAML1、FN1、Lumican、COL5A2、FCN1、TIMP1等；线粒体及能量提供相关基因：COX6C、COX8、MPP1、MRPL9、MRPL15、MRPL20、PMPCB、TIMM8B、ATP6J、ATP6M、ATP5H、ATP6H等；与类固醇激素合成相关基因：StAR、LOC51144、DHCR24、HSD11B1、HSD17B4、3β-HSD、enolase 1、SECRET等。
结论
1．经包含8300个基因位点的人表达谱基因芯片筛查，窦卵泡发育中期(直径5～12mm)的未成熟卵泡颗粒细胞和成熟卵泡颗粒细胞存在462个差异表达基因，说明人类卵母细胞发育、成熟过程受颗粒细胞所分泌的一系列物质时空上的调控密切相关。
2．对基因芯片结果比率不同的10个差异表达基因进行Real-time RT-PCR验证，除1个基因无显著差异外，其余均有显著差异，符合率达90％。提示本实验基因芯片的可信度，我们可以根据基因芯片的结果进行基因表达产物及其功能的进一步研究。
3．经基因芯片筛查未成熟卵泡颗粒细胞和成熟卵泡颗粒细胞存在190个基因上调表达，272个基因下调表达。说明随着卵泡的发育、成熟，有更多的调节因子参与，颗粒细胞在卵泡成熟阶段所具有的功能更复杂。
4．分析未成熟卵泡和成熟卵泡颗粒细胞差异表达基因，提示不同卵泡发育阶段颗粒细胞所起的作用不同。未成熟卵泡颗粒细胞主要是以促进细胞增殖、雌激素分泌和缝隙连接等基因表达占优势；成熟卵泡颗粒细胞主要与孕激素分泌、信号转导、物质转运、细胞外基质和粘液分泌等细胞功能相关基因表达占优势。
5．基因芯片结果提示不同卵泡发育阶段颗粒细胞产生不尽相同的信号转导因子、生长因子、细胞周期调节因子、旁分泌因子和能量物质作用于卵母细胞，调节卵母细胞成熟，或接受卵母细胞分泌的物质发生功能上的改变。
6．芯片结果提示除了与既往研究相一致的在卵泡发育过程中的基因优势表达和功能外，尚有许多在卵泡发育、成熟过程中功能未明的基因有待进一步研究。对这些基因及其表达产物和功能的研究将有助于增加我们对卵泡／卵母细胞发育、成熟机理的全面了解。
三、人类卵子体外成熟——体外受精—胚胎移植—冷冻复苏
目的
观察取卵周期准备、卵泡直径、IVM培养液成分和培养时间对未成熟卵的获取率、成熟率、受精率、卵裂率、优质胚胎率、着床率、妊娠率和出生率及其胚胎冷冻复苏移植的影响。
方法
以2002年7月～2004年7月在本院生殖中心进行卵子体外成熟的不孕不育患者为研究对象。以自然周期+绒毛膜促性腺激素(hCG)，或单用尿促卵泡素(HMG)+hCG，或常规促性腺激素释放激素激动剂(GnRH-a)长方案促排卵周期由于有卵巢过度刺激倾向而改为卵子体外成熟(IVM)周期获取未成熟卵。取卵时最大卵泡直径10～13.5mm不等，分别以TCM199或人输卵管液(HTF)为基础培养液，分别加入10％人血清蛋白(PPF)或10％合成血清物质(SSS)，再添加0.075 IU／ml r-FSH+0.5IU／mL hCG+1μg／mlβE2于上述IVM培养液中，观察取卵周期准备、卵泡直径、IVM培养液成分对未成熟卵的获取率、成熟率、受精率、卵裂率、优质胚胎率、着床率、妊娠率和出生率及其胚胎冷冻复苏移植结局。
结果：
1．自然周期、单用尿促卵泡素或是控制性促排卵周期各有1例妊娠。
2．当取卵时卵泡大小不均一且最大卵泡直径≥12mm时，获卵率下降，受精率下降、胚胎质量下降，无妊娠发生。3例妊娠均发生于卵泡发育大小较一致且最大卵泡直径＜12mm的取卵周期。
3．TCM199或人输卵管液(HTF)为基础培养液的2种不同的培养方案，卵子成熟率、受精率、卵裂率均差异无显著性，但优质胚胎的形成率前组显著高于后组(P＜0.01)。
4．IVM胚胎经冷冻复苏，胚胎移植能获得与常规IVF周期相似的妊娠成功，出生后代健康无畸形。
结论：
1．IVM周期取卵时卵泡大小不均一且最大卵泡直径≥12mm时，受精率和胚胎质量下降，将影响胚胎着床率和累积妊娠率。
2．以TCM199为基础培养液能提高未成熟卵体外成熟后受精卵所形成胚胎的发育能力和着床率，但仍低于常规IVF周期的着床率。
3．IVM胚胎经冷冻复苏，胚胎移植能获得与常规IVF周期相似的妊娠成功，出生后代健康无畸形。
Background:
Knowledge of the mechanisms involved in the oocytes and follicles development and maturation is essential to fully understand the human reproductive processes. Since 1930s, with the development of electron microscopy and molecular biological techniques, researches on germinal cells have advanced to the levels of ultra-structure and biological molecules such as DNA, mRNA and protein. These improvements have provided powerful technical platform to research the origin and differentiation of germinal cells. Furthermore, it has also give us opportunities to identify the mechanisms underlying the stimulation the primordial follicles from the quiescent to the growth phase, morphologically changes from primary follicle to secondary follicle then to mature follicle, and the oocytes and follicular developmental environment associated with the endocrinal, autocrinal and paracrinal actions. Most important, the birth of the first in vitro fertilization and embryo transfer (IVF-ET) baby in 1978 enhanced profound researches on human reproduction; furthermore, the development of genetic molecular techniques combined with the emergence of DNA array techniques, provided more powerful technical supports to research the mechanisms involved in follicular development and oocytes maturation.
To date, the implantation rate per cycle has still been close to 20%. In addition, only approximately 30%- 40% of the clinical pregnancy rate per IVF cycle and approximately 25% of living birth rate per IVF cycle have been observed in women who received assisted reproductive techniques. One of the important reasons is known to be the abnormal oocytes collected in the superovulatary environment during the controlled ovarian hyperstimulation procedure, resulting in low fertilization rate and poor quality embryo. On the other hand,
since the availability of in vitro maturation (IVM) of oocytes in 1983, IVM has been becoming interesting alternatives to classical assisted reproductive technology approaches, especially in those at high risk for ovarian hyperstimulation syndrome (OHSS). More than for their clinical and biological indications, IVM of oocytes can also be considered as good social and economic alternatives to the classical IVF treatment, based on their financial cost-effectiveness with exclusion of expensive medications. However, due to the asynchronous development of the nucleus and cytoplasm of immature oocytes found in the IVM program, the clinical application of IVM in the recent 20 years has still been limited. Until 2005, there have been only 300 IVM babies. Thus, deeper knowledge of the biological and pathological mechanisms should improve poor developmental potential of in vitro maturation oocytes and the quality of derived embryos, their successful implantation, maintenance of pregnancy, and offspring health.
Previous studies have shown that some ovarian factors are involved in the process of oocyte maturation. Researches had tried to add these factors to the medium to improve the outcome of oocyte matured in vitro; however, such IVM recipes are not comparable to physiological environment. Up to thousands of molecules are the process of follicle growth and oocyte maturation. Up to now, the interplay of growth factors and cytokines secreted by oocytes, cumulus cells and granulosa cells are unclear. Moreover, few studies work on the effects of hyperstimulation on ultrastructure of oocyte-cumulus complex (OCCs). Thus, we investigated the gene expression difference of granulosa cells collected from mature follicles and immature follicles and the ultrastructural changes of OCCs. These studies help to reveal the mechanisms of follicle growth and oocyte maturation, and are important for improvement of IVM culture system.
Part I Changes in the ultrastructure of human oocyte cumulus complexes at different follicular developmental environment and stages
Objectives
To examine the ultrastructural changes of of oocyte cumulus complexes (OCCs) when they matured in in vivo or in vitro environment, compare the ultrastructural differences of OCCs in natural status and conventional hyperstimulation treatment cycle, and investigate the effects of different cultural environment on the quality of oocytes. Methods The oocytes at different stages (GV, MI, and MII stages) were distinguished under light
microscopy. Samples (donated OCCs) were collected in natural cycles during laparoscopy operation, IVF/ICSI cycles, and IVM cycles. The ultrastructures of immatured oocytes in natural cycles and IVF/ICSI cycles, matured oocytes in IVF/ICSI cycles and IVM cycles were studied by transmission-electron microscopy. Results
The difference in the structure and distribution of cumulus cells, zona pellucida, microvilli, cortical granule, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, lipoid drop vesicles and centrioles was observed in OCCs that were developed in different developmental environment and were at different developmental stages.
1. Difference of the ultrastructures was observed in the GV stage OCCs which were collected in natural cycle and conventional ovarian hyperstimulation cycle. In OCCs of natural cycle, there were abundant lipoid drop vesicles and Golgi apparatus, no perivitelline space, dense and thin zona pellucida, and clear nucleoli. Each oocyte and its surrounding cumulus cells share a communication system, the gap junction, which embraced these cells as an intact functional system. The gap junction turned to be loose between the oocyte and its surrounding cumulus cells with no structure space at GV stage in IVF/ICSI treatment cycle. The number of cortical granule in oocyte increased; microvilli were long and thin and protruded into zona pellucida; the number of lipoid drops decreased, the membrane of nucleoli was less arranged. These changes suggested that the follicles were luteinized earlier.
2. Abundant organelles such as mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus, large lipoid drops were found in the cumulus cells of OCCs at MI stage in IVF/ICSI treatment cycle. Occasionally medulla-like body was detected. Cellular organelles and cortical granules were increased in oocytes; organelles such as mitochondria and lysosomes migrated toward cortical region gradually; a perivitelline space between cytoplasm membrane and zona pellucida emerged with vesicular structures; the nucleus disappeared; the thickness of zona pellucida increased to 11～12 um; and the microvilli became longer and protruded into zona pellucida.
3. A large mount of vesicular structures were distributed in the cytoplasm of matured oocytes at MII stage in IVF/ICSI cycle; the zona pellucida became wider and looser; the perivitelline space appeared and turned to spread, especially at the site of first pole body expelling; the microvilli retracted from the zona pellucida; the number of cortical granules increased; cortical granules migrated toward the margin of cells and arranged along oolemma linearly; the number of rough endoplasmic reticulum and Golgi apparatus
decreased; a "transparent" area with no organelle appeared in the centre of the oocyte; the number and structure of mitochondria were not changed. On the other hand. the number of mitochondria in the IVM oocytes decreased and the structure became less arranged; smooth endoplasmic reticulum with vesicular structures could be observed: the number of cortical granule decreased; mitochondria in the cumulus cells became swollen; some nuclei in granulosa cells disappeared; apoptosis happened in some granulosa cells. Conclusions
1. The GV stage OCCs collected from IVF/ICSI cycles, which mainly generated mature oocytes, had loose junction between oocytes and cumulus cells due to the overdose of FSH and hCG. Thus, nutrition transported through the gap junction to the oocytes was depleted. The impairment of communication between oocytes and cumulus cells and earlier distribution of cortical granules probably lead to decrease of fertilization rate and developmental potentials. The mature capacity of these oocytes was impaired by hyperstimulation hormone environment. Thus, the GV oocytes from IVF ICSI cycles were not suitable for further culture and embryo transplantation.
2. The cumulus cells surrounding oocytes at MI stage in hyperstimulation cycle showed active proliferation and function; the changes of zona pellucida and cortical granule of oocytes were fully ready to undergo further development.
3. The smooth endoplasmic reticulum was swollen in IVM oocytes, while the number of mitochondria decreased and their structure became less arranged; lipoid drops and cortical granules decreased. The cumulus cells showed obvious signs of apoptosis. It indicated that these changes were responsible for the poor quality of oocytes and impairment of developmental potential.
Part II The different expressions of gene profile of granulose cell from mature ovarian follicles and immature ovarian follicles
Objectives
To examine the gene expression profiles of granulose cells isolated from mature and immature ovarian follicles, and to attempt an understanding of the underlying molecular mechanism of granulosa cells involved in the maturation of ovarian follicle/oocyte.
Methods
The granulosa cells were isolated from the immature follicles in IVM cycle and mature follicles in conventional ovarian stimulation cycle. Total RNA was isolate and purify from granulosa cells. The granulosa cell from immature follicles were regarded as study group, while the granulosa cells from matured ones were regarded as control group. The expressive difference of study group and control group was investigated with the oligonucleotide microarray containing 8300 human genes. Ten of the genes were further confirmed by real-time RT PCR for their expression characteristics in granulosa cells.
Results
1. Changes in patterns of 462 genes expression in granulose cells were observed between mature and immature follicles. Among them. the expressions of 190 genes were up-regulated and 270 genes were down-regulated in granulose cells of immature follicles compared with mature ones. These genes included oncogene (15), ion channel and transport proteins (13), cellular cyclic protein (15). stress reaction protein (1) , proteins responsible for cellular structure and movement (18), cellular apoptosis related protein (4), DNA synthesis and repair, reconstruction protein (8), DNA binding proteins and transcription factors (15), cellular receptors (11), immunological related protein (20), cellular signal and transduction protein (54), proteins involved in metabolism (66), factors responsible for protein translation and synthesis (35), development related proteins (9), and others with unknown function (92). Some of the genes have 2 or more than 2 types of functions. 166 genes were remained to be unclassified.
2. According to the rate of up-regulated genes and down-regulated genes, the expression ration of 6 down-regulated genes (STAR, MPHOSPH6, CALM1, MAML1, CD53, RRAGA) and 4 up-regulated genes (ICAM1、 CYR61、 ZNF264、 STMN1) were selected and further confirmed by real-time RT-PCR. The ratio of difference gene expression examined by microarray of the 6 down-regulated genes were 0.16、 0.219、 0.214、 0.319、 0.183 and 0.411, respectively; while the ratio by real-time RT-PCR were 0.166、 0.086、 0.213、 0.186、 0.177 and 0.345, respectively. Furthermore, the ratio of difference gene expression examined by microarray of the 4 up-regulated genes were 2.385、 3.619、 3.992 and 10.245, respectively; while the ratio by real-time RT-PCR were2.28、 2.023、 2.30 and 5.07, respectively. The trend of the differences of gene expression between microarray and real-time RT-PCR was similar. The difference of gene expression between the two groups of these ten genes was statistically analyzed, resulting in only gene with no significant change, while the other genes being significantly changed between the two groups
(P<0.001).
3. The microarray results showed that genes related to proliferation, mitochondria duplication, gap junction, estrogen secretion, energy synthesis were up-regulated in the granulosa cells from immature follicles. These up-regulated genes included: growth factors: CTGF、 TIEG、 PDGFC、 FGFR-1、 CGR19, et al; cell cycle modulation protein and apoptosis related factors: SMC1L1、 HMG2、 PTP4A1、 cyclin B、 CDK9、 STMN1、 melanoma antigen, et al; genes related to signaling transduction and gap junction: PRSS11、 PTP4A1、 CYR61、 GAB1、 ICAM1 (CD54)、 Integrins -β8、 connexin 43, et al. genes related to cystoskeleton, such as microtubule protein were also dominant in expression: XPO1、 GPAT、 SPP1、 LOC51174、 MIP-T3, et al. Another group of up-regulated genes were modulation factors stimulating estrogen secretion: CYP19、 IRS1 、 ADRBK2、 CRHBP, et al.
4. The microarray results showed that genes related to steroid synthesis, mitochondria synthesis, energy transportation, cellular signaling transduction, extracellular matrix formation, mucus secretion were up-regulated in the granulosa cells from mature follicles. These genes included: genes related to signaling transduction: PTPRG、 PRKCD、 KIAA010、 FDPS、 IMPA1、 TPI1、 PSPHL、 PIP5K2B, et al; genes related to extracellular matrix formation and mucus secretion: PTX3、 CALM1、 MAML1、 FN1、 Lumican、 COL5A2、 FCN1、 TIMP1, et al; genes related to mitochondria and energy supply: COX6C、 COX8、 MPP1、 MRPL9、 MRPL15、 MRPL20、 PMPCB、 TIMM8B、 ATP6J、 ATP6M、 ATP5H、 ATP6H, et al; genes related to steroid synthesis: StAR、 LOC51144、 DHCR24、 HSD11B1、 HSD17B4、 3β-HSD、 enolase 1、 SECRET, et al.
Conclusions
1. The expression difference of 462 genes between the granulosa cells from immature and mature follicles was detected by microarray. It suggests that the growth and mature of human oocytes are modulated by factors secreted by granulosa cells of follicles at different developmental stages.
2. Ten of the genes showing expression difference were confirmed by real-time RT-PCR. Except one gene showed no difference by real-time RT-PCR, the real-time RT-PCR results of other gene were consistent with results of microarray. It suggested that the results of microarray are reliable. We can further investigate the gene expression pattern and do some functional assay according to the results of microarray.
3. The expressions of 190 up-regulated genes and 270 down-regulated genes were observed in granulose cells from immatured follicles compared with matured follicles, suggesting that more regulatory factors are involved in the developmental process as the follicles grew
and matured, and the function of granulose cells of these follicles turn to be more complex.
4. We hypothesize that the roles of granulose cells at different growth stages were different. During early stage of follicular development, the granulose cells are mainly responsible for cellular proliferation, estrogen secretion and gap junction; while follicles became more mature, the granulose cells transfer to mediate progestogen secretion, signal transduction, substance transportation, extracellular matrix formation and mucus secretion.
5. The results of microarray indicated that the signaling transduction factors, growth factors, cell cycle modulation factors and paracrine factors secreted by granulosa cells are somewhat different for follicles at different stages. These factors modulate oocyte maturation, or in turn, the functions of granulosa cells are modified by the secretion from oocytes.
6. Although the results of microarray suggested us dominant gene expression during the process of follicle development, the detailed function of the genes are remained to be investigated. Further study of these genes will lead to better understanding of the mechanisms of oocyte/follicle growth and maturation.
Part III In vitro maturation—in vitro fertilization—embryo transfer—frozen-thawed of human oocytes
Objectives
To investigate the influence of ovarian follicle diameter, IVM culture media and culture duration on the number of retrieved immature oocytes, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed germinal vesicle stage and in vitro matured human oocytes. Methods
The oocytes were obtained by follicular aspiration from follicle-stimulating hormone-treated women undergoing oocyte retrieval for in vitro maturation between July 2002 and July 2004. IVM program was performed due to the possible risk of ovarian hyperstimulation in IVF-ET program. All the oocytes retrieved from follicles with 10-13.5 mm in diameter were allowed to remain in medium M-199 (TCM 199) or HTF supplemented with 10% human
plasma protein or 10% synthesized serum substance, and 0.075 IU/ml r-FSH +0.51U/mL hCG+1μg/mlβE2. Then the effects of follicle diameter, IVM culture medium and culture duration on the retrieval rate, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, clinical pregnancy rate, delivery rate and outcome of derived embryos transfer of in vitro matured human oocytes was investigated. Results
1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;
2. Using different culture medium (TCM 199 and HTF) had no effects on the maturation rate, fertilization rate, and cleavage rate of oocytes, however, the high-quality embryos formation rate was higher for the oocytes cultured in HTF medium (P <0.01) .
3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.
Conclusions
1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm. the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;
2. Although TCM199-based medium could improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes, the implantation rate of these embryos is still lower than the implantation rate of embryos generated in conventional IVF or ICSI cycles.
3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.
对卵母细胞和卵泡发育、成熟机制的研究是人类揭示自身繁衍的奥秘、提高人口素质的需要。自从20世纪30年代以来，随着物理化学技术，尤其是电子显微镜技术的发明和分子生物学技术的不断发展，打破了光学显微研究细胞的局限而进入了超微结构和分子生物水平的领域，为包括生殖细胞的来源、原始生殖细胞分化、始基卵泡由静止池进入生长发育阶段的动因、初级卵泡发育为次级卵泡和成熟卵泡过程的形态学变化规律和内分泌环境、白分泌和旁分泌作用等研究提供了技术平台。尤其是1978年世界首例体外受精—胚胎移植(in vitro fertilization-embryo transfer，ⅣF-ET)婴儿的诞生，一方面为人类生殖机制的深入研究提供了契机，另一方面更激起了生殖学者对人类生殖机制了解的迫切需求，同期遗传分子生物学技术的迅猛发展、生物芯片技术的出现，更为我们探索生命发生、进化、生长、发育、凋亡的规律提供了有力的技术支撑。
目前ⅣF周期的胚胎着床率大约在20％左右，妊娠率在30％～40％，活胎出生率在25％左右，主要原因之一在于促排卵周期所获卵子有相当一部分发育异常，不能受精或不能形成高质量的胚胎。1983年取得的卵母细胞体外成熟(ⅣM)的成功，由于不需要促排卵，简化了ⅣF程序、节约了治疗费用、避免了ⅣF周期严重卵巢过度刺激综合症的发生，因而受到了人们的关注。然而，20多年来，ⅣM技术的应用一直有限，据2005年统计仅出生子代300名，原因在于ⅣM技术至今未克服卵母细胞体外成熟过程中胞核和胞浆成熟不一致的难题，对卵母细胞发育、受精、胚胎发育起关键作用的胞浆成熟远远落后于胞核。对卵母细胞／卵泡发育、成熟机制和胚胎发育机制的研究，将有助于提高辅助生育技术(ART)的成功率和子代健康状况。从长远来说有助于我们对人类生殖繁衍奥秘的了解，为妇女生殖健康措施制定提供理论指导。
卵母细胞在卵巢中进行减数分裂的过程称为卵的发生，这个过程包括增殖期、生长期以及成熟期3个阶段。卵泡的发生、发育和成熟包括卵泡的募集、选择和优势化。卵母细胞的成熟与其周围的卵泡细胞的作用密不可分，卵母细胞发育伴随了卵泡的增大，当卵泡发育到一定大小时才发生卵母细胞核／质的成熟。卵泡发育根据形态和功能可分为以下3个阶段：(1)静止卵泡：包括原始卵泡、中间卵泡和初级卵泡；(2)早期生长卵泡：包括次级卵泡、窦前卵泡、早期窦卵泡、选择卵泡；(3)窦卵泡：直径达2mm，对FSH敏感性增加，依赖FSH继续发育，继后受LH峰作用最终成熟。
静止卵泡是非激素依赖性，受遗传因素和局部调节因子的影响，进行分化、增殖。从始基卵泡发育到次级卵泡机制不清，1991年Horie等证实由颗粒细胞产生的Kit Ligand(KL)作用于卵泡膜间质细胞的相应受体(c-Kit受体)，在无Gn的情况下单独刺激静止卵泡进入生长发育期，窦前卵泡的卵母细胞直径100～130μm，已达成熟期大小，直径2mm的窦卵泡颗粒细胞FSH受体充足，已具备了体外成熟的条件。从2mm到18mm需要25天时间，后15天相当于月经周期的卵泡期。窦卵泡生长至直径10-14mm时卵母细胞重新启动减数分裂，但仍停留在生殖泡期，发育至排卵前卵泡，直径约为15-25mm时受促黄体生成素(LH)作用，卵泡发生生殖泡破裂(GVBD)，并成为成熟卵(MⅡ)，发生排卵。卵泡进入生长状态(次级卵泡2层GC出现)至发育成熟共需85天时间。
处于MⅡ期的卵母细胞光镜下直径约150μm，从外到内，由透明带(ZP)、卵周间隙、卵黄膜、卵浆(亦称卵母细胞皮质)、细胞核及核仁组成。卵黄膜与透明带之间为充满液体的卵周间隙，内见圆形第一极体。胞浆匀称，浅灰色，颗粒均匀。当卵母细胞形态异常、胞质分布异常以及第一极体形态发生改变时将导致受精能力和进一步胚胎发育能力的下降，但这种改变伴随着的超微结构改变尚不清楚。
卵母细胞的大多数细胞器最初分布于近核区，随着卵母细胞生长，线粒体、高尔基体、粗面内质网、皮质颗粒等细胞器不断丰富和增多，并逐渐向皮质区迁移，在细胞中央形成不含细胞器的“透明区”，卵母细胞成熟后，皮质颗粒排列在质膜下，皮质颗粒的大量增加和迁移并沿质膜下呈线性排列是卵母细胞胞质成熟的一个重要标志。从初级卵母细胞到MⅡ期卵母细胞，其胞膜表面微绒毛的数量不断增多，微绒毛长度逐渐增加，至完全成熟后微绒毛数量下降。
在卵泡发育过程中卵泡细胞线粒体和粗面内质网丰富，颗粒细胞和卵母细胞伸出突起穿过透明带相互联系。卵母细胞、卵丘细胞和卵泡细胞之间存在广泛的缝隙连接形成一个完整的功能联合体，这种结构一直至卵母细胞恢复减数分裂前才发生松解。
在卵泡／卵母细胞的发育过程中卵巢局部的微环境参与了整个过程，有研究发现卵泡颗粒细胞产生的抑制素A、抑制素B和激活素A是卵母细胞成熟基本细胞因子，胰岛素样生长因子Ⅰ／Ⅱ、表皮生长因子、转化生长因子、骨形成蛋白、生长与分化因了、肿瘤坏死因子、白介素、抗苗勒管激素、卵细胞成熟抑制因子、有丝分裂原激活蛋白激酶、促成熟因子、连接蛋白43、生长与分化激素、妊娠相关血浆蛋白a、血管上皮生长因子、磷脂酰肌醇3激酶(PI3K)等对卵母细胞／卵泡发育的启动、生长、成熟起着极为重要的作用，当上述物质缺失或编码基因突变将导致卵细胞或卵泡发育、成熟障碍。
虽然已明确了卵泡发育的基本形态学改变和FSH、LH、T、E_2等激素的周期性变化，并对影响卵母细胞成熟的卵巢内微环境因素的研究取得了一定的进展，尝试在卵母细胞体外成熟培养中添加上述的某些物质，但仍未达到生理状态的成熟。卵泡／卵母细胞发生、发育、成熟过程极为复杂，包含其中的分子可能有数十种、数百种、甚至数千种?由于卵泡壁层颗粒细胞、膜细胞、卵丘颗粒细胞和卵母细胞之间众多生长因子、细胞因子的相互调节作用机制仍未完全清楚，对于ART及其衍生技术的不断涌现，卵泡和卵母细胞受超生理剂量的促性激素和性激素的影响以及在体外培养过程中的一系列超微结构的形态学变化更缺乏了解。因此，我们试图从未成熟卵泡和成熟卵泡颗粒细胞基因表达差异、卵细胞超微结构形态的改变研究卵母细胞、卵泡生长、发育、成熟机制，分析参与卵母细胞／卵泡成熟的相关因素，获取卵母细胞成熟的相关信息，为建立合理的ⅣM体系提供理论支持，探索未成熟卵获取周期准备方案、取卵时机和体外培养条件。
一、人未成熟卵和成熟卵卵丘复合物超微结构研究
目的
观察人卵丘复合物(oocyte-cumulus complex，OCCs)在体内和体外从未成熟发育至成熟的超微结构变化，并比较自然状态和常规促排卵ⅣF／ICSI周期未成熟OCCs超微结构的区别，分析不同条件和环境对卵细胞质量的影响。
方法：
用光镜分辨生发泡期(GⅤ期)、生发池破裂期(MⅠ期)的未成熟卵和达到MⅡ期的成熟卵。标本来自妇科腹腔镜手术的自然周期、ⅣF／ICSI周期、ⅣM周期捐赠的卵丘复合物(OCCs)。对妇科腹腔镜手术的自然周期未成熟卵(GⅤ期)、促排卵周期未成熟卵(GⅤ／MⅠ)、促排卵周期体内成熟卵(MⅡ)、体外成熟培养成熟卵(MⅡ)各3枚，分别经透射电镜技术处理，观察OCCs的超微结构。
结果
不同发育条件、发育阶段人卵丘颗粒细胞发育、卵母细胞透明带、微绒毛、皮质颗粒、线粒体结构、溶酶体、内质网、高尔基体、脂滴空泡和中心体等结构和分布方面均有差异。
1．自然周期的GⅤ期卵与常规促排卵ⅣF／ICSI周期获取的GⅤ期OCCs在超微结构方面存在显著差异。自然周期GⅤ期卵有丰富的脂滴、空泡，核仁清晰可见，无卵周间隙，透明带致密、较薄，高尔基体丰富，卵丘细胞与卵丘细胞之间以及卵丘细胞与卵母细胞之间存在广泛的缝隙连接，为一完整的功能合胞体。常规促排卵ⅣF／ICSI周期的GⅤ期卵丘细胞与卵母细胞连接松散，出现无结构的间隙，颗粒细胞染色质边聚于核膜等凋亡改变，卵母细胞皮质颗粒略有增多，微绒毛细长而竖立伸入透明带，脂滴减少，核膜皱褶出现，已出现受LH作用的改变。
2．ⅣF／ICSI周期体内未成熟卵母细胞(MⅠ期)卵丘细胞粗面内质网、线粒体、高尔基体、溶酶体、微管及大颗粒脂滴等细胞器极为丰富，偶见髓样小体；卵母细胞细胞器增多，线粒体、溶酶体、等结构逐渐移向卵母细胞皮质区，卵母细胞内皮质颗粒增多，质膜边缘略有增加；卵母细胞膜与透明带间出现卵周间隙，内见泡状结构；卵细胞胞核消失；透明带明显增厚，约为10～12μm，结构松散；微绒毛增粗，竖立、伸入透明带。
3．ⅣF／ICSI周期体内成熟卵母细胞(MⅡ期)胞质内为散在分布数目较多的细小空泡状结构，透明带逐渐增宽、松散；卵周间隙出现、增宽，以第一极体排出处最明显，微绒毛自透明带回缩，皮质颗粒丰富，向细胞边缘聚集，并呈线状排列在卵膜边缘；粗面内质网和高尔基体减少，在细胞中央出现不含细胞器的“透明区”；线粒体减少不明显、结构仍然清晰可见；而体外成熟卵母细胞线粒体数目减少，结构模糊，并出现较多泡状结构的滑面内质网，皮质颗粒较少；卵丘细胞线粒体肿胀、嵴消失，染色质发生异质化、边缘化，部分颗粒细胞核消失、细胞变型、质膜不完整等凋亡改变明显。
结论
1．经常规促排卵的IVF／ICSI周期，在大部分卵母细胞成熟的情况下产生的GV期卵丘复合物，由于受超生理剂量FSH以及hCG的影响，卵丘细胞与卵母细胞连接松散，将导致卵母细胞过早缺乏颗粒细胞通过缝隙连接输送的营养以及卵母细胞皮质颗粒过早出现，可能是受精率和胚胎发育能力下降的原因。超生理状态的激素坏境将影响发育延迟卵母细胞体外成熟的能力。因此，来自于常规促排卵周期的GV卵不适合进一步的培养，用于胚胎移植。
2．促排卵周期MI期卵丘细胞呈旺盛的增殖和功能活动状态，卵母细胞内的胞质结构、皮质颗粒、透明带等已发生了适合于卵母细胞成熟的显著的形态学改变，为卵母细胞成熟做好的充分的准备，具有进一步发育的潜能。
3．经体外成熟母细胞滑面内质网扩张，线粒体数目减少、结构模糊，脂滴减少，皮质颗粒减少，而且卵丘颗粒细胞凋亡改变明显，可能是卵母细胞质量下降、使其进一步发育能力受损的原因。
二、人类未成熟卵泡和成熟卵泡颗粒细胞基因表达差异
目的
分析人未成熟卵泡和成熟卵泡颗粒细胞基因表达差异，探讨卵泡颗粒细胞在卵泡／卵母细胞成熟过程中可能参与的分子机制。
方法
获取IVM周期未成熟卵泡(直径5～12mm)的卵泡液和常规促排卵IVF周期成熟卵(直径≥14mm)的卵泡液，分离颗粒细胞，抽提、纯化mRNA。将未成熟卵泡颗粒细胞设为研究组，成熟卵泡颗粒细胞设为对照组，采用含8300个人类基因的基因芯片进行差异性表达研究，并对其中有差异性表达的10个基因采用荧光实时定量技术(Real-time RT-PCR)进行验证。
结果
1．经基因芯片筛查，人未成熟卵泡和成熟卵泡颗粒细胞比较有462个基因表达存在差异，其中190个基因为上调表达，272个基因为下调表达。对这462个差异表达基因进行分类：原癌基因和抑癌基因15个；离子通道和运输蛋白13个；细胞周期蛋白类15个；外压反应蛋白1个；细胞骨架和运动蛋白18个；细胞凋亡相关的蛋白4个；DNA合成和修复、重组蛋白8个；DNA结合、转录和转录因子15个；细胞受体蛋白11个；免疫相关蛋白20个；细胞信号和传递蛋白54个；代谢相关调节蛋白66个；蛋白翻译合成调节因子36个；发育相关蛋白9个；其它蛋白92个，其中某些基因兼有2种或2种以上功能。尚有166个基因未分类或功能未明。
2．应用Real-time RT-PCR方法，按下调和上调表达基因的比例，分别选取其中6个下调基因和4个上调基因进行验证。6个下调基因STAR、MPHOSPH6、CALM1、MAML1、CD53和RRAGA基因芯片比率分别为0.16、0.219、0.214、0.319、0.183和0.411，Real-time RT-PCR的比率分别为0.166、0.086、0.213、0.186、0.177、0.345；4个上调基因ICAM1、CYR61、ZNF264和STMN1基因芯片的比率分别为2.385、3.619、3.992和10.245，Real-time RT-PCR的比率分别为2.28、2.023、2.30和5.07，比率趋势两者相符。10个基因的Real-time RT-PCR扩增产物经统计学分析，ICAM1表达无差异(P=0.11)，其他9个基因表达均存在显著差异(P＜0.001)。
3．排卵前未成熟卵颗粒细胞的基因表达分析，提示以调节颗粒细胞增殖、线粒体复制、细胞间缝隙连接、雌激素分泌、能量合成的基因上调表达为主。生长因子类如：CTGF、TIEG、PDGFC、FGFR-1、CGR19等；周期调节蛋白及凋亡相关因子如：SMC1L1、HMG2，PTP4A(?)、cyclin B、CDK9、STMN1、melanoma antigen等；与信息转导和细胞连接相关的基因：PRSS11、PTP4A1、CYR61、GAB1、ICAM1(CD54)、Integrins-β8、connexin 43等。与细胞骨架形成有关的微管蛋白等也有优势表达，如XPO1、GPAT、SPP1、LOC51174、MIP-T3等。与刺激雌激素合成相关的调节因子如CYP19、IRS1、ADRBK2、CRHBP等。
4．成熟卵颗粒细胞基因表达分析，提示以调节类固醇激素合成、线粒体合成的能量转运、细胞信息转导、细胞外基质形成和粘液分泌的基因上调表达为主。与信号转导相关的基因如：PTPRG、PRKCD、KIAA010、FDPS、IMPA1、TPI1、PSPHL、PIP5K2B等；细胞外基质形成、粘液分泌相关的基因：PTX3、CALM1、MAML1、FN1、Lumican、COL5A2、FCN1、TIMP1等；线粒体及能量提供相关基因：COX6C、COX8、MPP1、MRPL9、MRPL15、MRPL20、PMPCB、TIMM8B、ATP6J、ATP6M、ATP5H、ATP6H等；与类固醇激素合成相关基因：StAR、LOC51144、DHCR24、HSD11B1、HSD17B4、3β-HSD、enolase 1、SECRET等。
结论
1．经包含8300个基因位点的人表达谱基因芯片筛查，窦卵泡发育中期(直径5～12mm)的未成熟卵泡颗粒细胞和成熟卵泡颗粒细胞存在462个差异表达基因，说明人类卵母细胞发育、成熟过程受颗粒细胞所分泌的一系列物质时空上的调控密切相关。
2．对基因芯片结果比率不同的10个差异表达基因进行Real-time RT-PCR验证，除1个基因无显著差异外，其余均有显著差异，符合率达90％。提示本实验基因芯片的可信度，我们可以根据基因芯片的结果进行基因表达产物及其功能的进一步研究。
3．经基因芯片筛查未成熟卵泡颗粒细胞和成熟卵泡颗粒细胞存在190个基因上调表达，272个基因下调表达。说明随着卵泡的发育、成熟，有更多的调节因子参与，颗粒细胞在卵泡成熟阶段所具有的功能更复杂。
4．分析未成熟卵泡和成熟卵泡颗粒细胞差异表达基因，提示不同卵泡发育阶段颗粒细胞所起的作用不同。未成熟卵泡颗粒细胞主要是以促进细胞增殖、雌激素分泌和缝隙连接等基因表达占优势；成熟卵泡颗粒细胞主要与孕激素分泌、信号转导、物质转运、细胞外基质和粘液分泌等细胞功能相关基因表达占优势。
5．基因芯片结果提示不同卵泡发育阶段颗粒细胞产生不尽相同的信号转导因子、生长因子、细胞周期调节因子、旁分泌因子和能量物质作用于卵母细胞，调节卵母细胞成熟，或接受卵母细胞分泌的物质发生功能上的改变。
6．芯片结果提示除了与既往研究相一致的在卵泡发育过程中的基因优势表达和功能外，尚有许多在卵泡发育、成熟过程中功能未明的基因有待进一步研究。对这些基因及其表达产物和功能的研究将有助于增加我们对卵泡／卵母细胞发育、成熟机理的全面了解。
三、人类卵子体外成熟——体外受精—胚胎移植—冷冻复苏
目的
观察取卵周期准备、卵泡直径、IVM培养液成分和培养时间对未成熟卵的获取率、成熟率、受精率、卵裂率、优质胚胎率、着床率、妊娠率和出生率及其胚胎冷冻复苏移植的影响。
方法
以2002年7月～2004年7月在本院生殖中心进行卵子体外成熟的不孕不育患者为研究对象。以自然周期+绒毛膜促性腺激素(hCG)，或单用尿促卵泡素(HMG)+hCG，或常规促性腺激素释放激素激动剂(GnRH-a)长方案促排卵周期由于有卵巢过度刺激倾向而改为卵子体外成熟(IVM)周期获取未成熟卵。取卵时最大卵泡直径10～13.5mm不等，分别以TCM199或人输卵管液(HTF)为基础培养液，分别加入10％人血清蛋白(PPF)或10％合成血清物质(SSS)，再添加0.075 IU／ml r-FSH+0.5IU／mL hCG+1μg／mlβE2于上述IVM培养液中，观察取卵周期准备、卵泡直径、IVM培养液成分对未成熟卵的获取率、成熟率、受精率、卵裂率、优质胚胎率、着床率、妊娠率和出生率及其胚胎冷冻复苏移植结局。
结果：
1．自然周期、单用尿促卵泡素或是控制性促排卵周期各有1例妊娠。
2．当取卵时卵泡大小不均一且最大卵泡直径≥12mm时，获卵率下降，受精率下降、胚胎质量下降，无妊娠发生。3例妊娠均发生于卵泡发育大小较一致且最大卵泡直径＜12mm的取卵周期。
3．TCM199或人输卵管液(HTF)为基础培养液的2种不同的培养方案，卵子成熟率、受精率、卵裂率均差异无显著性，但优质胚胎的形成率前组显著高于后组(P＜0.01)。
4．IVM胚胎经冷冻复苏，胚胎移植能获得与常规IVF周期相似的妊娠成功，出生后代健康无畸形。
结论：
1．IVM周期取卵时卵泡大小不均一且最大卵泡直径≥12mm时，受精率和胚胎质量下降，将影响胚胎着床率和累积妊娠率。
2．以TCM199为基础培养液能提高未成熟卵体外成熟后受精卵所形成胚胎的发育能力和着床率，但仍低于常规IVF周期的着床率。
3．IVM胚胎经冷冻复苏，胚胎移植能获得与常规IVF周期相似的妊娠成功，出生后代健康无畸形。
Background:
Knowledge of the mechanisms involved in the oocytes and follicles development and maturation is essential to fully understand the human reproductive processes. Since 1930s, with the development of electron microscopy and molecular biological techniques, researches on germinal cells have advanced to the levels of ultra-structure and biological molecules such as DNA, mRNA and protein. These improvements have provided powerful technical platform to research the origin and differentiation of germinal cells. Furthermore, it has also give us opportunities to identify the mechanisms underlying the stimulation the primordial follicles from the quiescent to the growth phase, morphologically changes from primary follicle to secondary follicle then to mature follicle, and the oocytes and follicular developmental environment associated with the endocrinal, autocrinal and paracrinal actions. Most important, the birth of the first in vitro fertilization and embryo transfer (IVF-ET) baby in 1978 enhanced profound researches on human reproduction; furthermore, the development of genetic molecular techniques combined with the emergence of DNA array techniques, provided more powerful technical supports to research the mechanisms involved in follicular development and oocytes maturation.
To date, the implantation rate per cycle has still been close to 20%. In addition, only approximately 30%- 40% of the clinical pregnancy rate per IVF cycle and approximately 25% of living birth rate per IVF cycle have been observed in women who received assisted reproductive techniques. One of the important reasons is known to be the abnormal oocytes collected in the superovulatary environment during the controlled ovarian hyperstimulation procedure, resulting in low fertilization rate and poor quality embryo. On the other hand,
since the availability of in vitro maturation (IVM) of oocytes in 1983, IVM has been becoming interesting alternatives to classical assisted reproductive technology approaches, especially in those at high risk for ovarian hyperstimulation syndrome (OHSS). More than for their clinical and biological indications, IVM of oocytes can also be considered as good social and economic alternatives to the classical IVF treatment, based on their financial cost-effectiveness with exclusion of expensive medications. However, due to the asynchronous development of the nucleus and cytoplasm of immature oocytes found in the IVM program, the clinical application of IVM in the recent 20 years has still been limited. Until 2005, there have been only 300 IVM babies. Thus, deeper knowledge of the biological and pathological mechanisms should improve poor developmental potential of in vitro maturation oocytes and the quality of derived embryos, their successful implantation, maintenance of pregnancy, and offspring health.
Previous studies have shown that some ovarian factors are involved in the process of oocyte maturation. Researches had tried to add these factors to the medium to improve the outcome of oocyte matured in vitro; however, such IVM recipes are not comparable to physiological environment. Up to thousands of molecules are the process of follicle growth and oocyte maturation. Up to now, the interplay of growth factors and cytokines secreted by oocytes, cumulus cells and granulosa cells are unclear. Moreover, few studies work on the effects of hyperstimulation on ultrastructure of oocyte-cumulus complex (OCCs). Thus, we investigated the gene expression difference of granulosa cells collected from mature follicles and immature follicles and the ultrastructural changes of OCCs. These studies help to reveal the mechanisms of follicle growth and oocyte maturation, and are important for improvement of IVM culture system.
Part I Changes in the ultrastructure of human oocyte cumulus complexes at different follicular developmental environment and stages
Objectives
To examine the ultrastructural changes of of oocyte cumulus complexes (OCCs) when they matured in in vivo or in vitro environment, compare the ultrastructural differences of OCCs in natural status and conventional hyperstimulation treatment cycle, and investigate the effects of different cultural environment on the quality of oocytes. Methods The oocytes at different stages (GV, MI, and MII stages) were distinguished under light
microscopy. Samples (donated OCCs) were collected in natural cycles during laparoscopy operation, IVF/ICSI cycles, and IVM cycles. The ultrastructures of immatured oocytes in natural cycles and IVF/ICSI cycles, matured oocytes in IVF/ICSI cycles and IVM cycles were studied by transmission-electron microscopy. Results
The difference in the structure and distribution of cumulus cells, zona pellucida, microvilli, cortical granule, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, lipoid drop vesicles and centrioles was observed in OCCs that were developed in different developmental environment and were at different developmental stages.
1. Difference of the ultrastructures was observed in the GV stage OCCs which were collected in natural cycle and conventional ovarian hyperstimulation cycle. In OCCs of natural cycle, there were abundant lipoid drop vesicles and Golgi apparatus, no perivitelline space, dense and thin zona pellucida, and clear nucleoli. Each oocyte and its surrounding cumulus cells share a communication system, the gap junction, which embraced these cells as an intact functional system. The gap junction turned to be loose between the oocyte and its surrounding cumulus cells with no structure space at GV stage in IVF/ICSI treatment cycle. The number of cortical granule in oocyte increased; microvilli were long and thin and protruded into zona pellucida; the number of lipoid drops decreased, the membrane of nucleoli was less arranged. These changes suggested that the follicles were luteinized earlier.
2. Abundant organelles such as mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus, large lipoid drops were found in the cumulus cells of OCCs at MI stage in IVF/ICSI treatment cycle. Occasionally medulla-like body was detected. Cellular organelles and cortical granules were increased in oocytes; organelles such as mitochondria and lysosomes migrated toward cortical region gradually; a perivitelline space between cytoplasm membrane and zona pellucida emerged with vesicular structures; the nucleus disappeared; the thickness of zona pellucida increased to 11～12 um; and the microvilli became longer and protruded into zona pellucida.
3. A large mount of vesicular structures were distributed in the cytoplasm of matured oocytes at MII stage in IVF/ICSI cycle; the zona pellucida became wider and looser; the perivitelline space appeared and turned to spread, especially at the site of first pole body expelling; the microvilli retracted from the zona pellucida; the number of cortical granules increased; cortical granules migrated toward the margin of cells and arranged along oolemma linearly; the number of rough endoplasmic reticulum and Golgi apparatus
decreased; a "transparent" area with no organelle appeared in the centre of the oocyte; the number and structure of mitochondria were not changed. On the other hand. the number of mitochondria in the IVM oocytes decreased and the structure became less arranged; smooth endoplasmic reticulum with vesicular structures could be observed: the number of cortical granule decreased; mitochondria in the cumulus cells became swollen; some nuclei in granulosa cells disappeared; apoptosis happened in some granulosa cells. Conclusions
1. The GV stage OCCs collected from IVF/ICSI cycles, which mainly generated mature oocytes, had loose junction between oocytes and cumulus cells due to the overdose of FSH and hCG. Thus, nutrition transported through the gap junction to the oocytes was depleted. The impairment of communication between oocytes and cumulus cells and earlier distribution of cortical granules probably lead to decrease of fertilization rate and developmental potentials. The mature capacity of these oocytes was impaired by hyperstimulation hormone environment. Thus, the GV oocytes from IVF ICSI cycles were not suitable for further culture and embryo transplantation.
2. The cumulus cells surrounding oocytes at MI stage in hyperstimulation cycle showed active proliferation and function; the changes of zona pellucida and cortical granule of oocytes were fully ready to undergo further development.
3. The smooth endoplasmic reticulum was swollen in IVM oocytes, while the number of mitochondria decreased and their structure became less arranged; lipoid drops and cortical granules decreased. The cumulus cells showed obvious signs of apoptosis. It indicated that these changes were responsible for the poor quality of oocytes and impairment of developmental potential.
Part II The different expressions of gene profile of granulose cell from mature ovarian follicles and immature ovarian follicles
Objectives
To examine the gene expression profiles of granulose cells isolated from mature and immature ovarian follicles, and to attempt an understanding of the underlying molecular mechanism of granulosa cells involved in the maturation of ovarian follicle/oocyte.
Methods
The granulosa cells were isolated from the immature follicles in IVM cycle and mature follicles in conventional ovarian stimulation cycle. Total RNA was isolate and purify from granulosa cells. The granulosa cell from immature follicles were regarded as study group, while the granulosa cells from matured ones were regarded as control group. The expressive difference of study group and control group was investigated with the oligonucleotide microarray containing 8300 human genes. Ten of the genes were further confirmed by real-time RT PCR for their expression characteristics in granulosa cells.
Results
1. Changes in patterns of 462 genes expression in granulose cells were observed between mature and immature follicles. Among them. the expressions of 190 genes were up-regulated and 270 genes were down-regulated in granulose cells of immature follicles compared with mature ones. These genes included oncogene (15), ion channel and transport proteins (13), cellular cyclic protein (15). stress reaction protein (1) , proteins responsible for cellular structure and movement (18), cellular apoptosis related protein (4), DNA synthesis and repair, reconstruction protein (8), DNA binding proteins and transcription factors (15), cellular receptors (11), immunological related protein (20), cellular signal and transduction protein (54), proteins involved in metabolism (66), factors responsible for protein translation and synthesis (35), development related proteins (9), and others with unknown function (92). Some of the genes have 2 or more than 2 types of functions. 166 genes were remained to be unclassified.
2. According to the rate of up-regulated genes and down-regulated genes, the expression ration of 6 down-regulated genes (STAR, MPHOSPH6, CALM1, MAML1, CD53, RRAGA) and 4 up-regulated genes (ICAM1、 CYR61、 ZNF264、 STMN1) were selected and further confirmed by real-time RT-PCR. The ratio of difference gene expression examined by microarray of the 6 down-regulated genes were 0.16、 0.219、 0.214、 0.319、 0.183 and 0.411, respectively; while the ratio by real-time RT-PCR were 0.166、 0.086、 0.213、 0.186、 0.177 and 0.345, respectively. Furthermore, the ratio of difference gene expression examined by microarray of the 4 up-regulated genes were 2.385、 3.619、 3.992 and 10.245, respectively; while the ratio by real-time RT-PCR were2.28、 2.023、 2.30 and 5.07, respectively. The trend of the differences of gene expression between microarray and real-time RT-PCR was similar. The difference of gene expression between the two groups of these ten genes was statistically analyzed, resulting in only gene with no significant change, while the other genes being significantly changed between the two groups
(P<0.001).
3. The microarray results showed that genes related to proliferation, mitochondria duplication, gap junction, estrogen secretion, energy synthesis were up-regulated in the granulosa cells from immature follicles. These up-regulated genes included: growth factors: CTGF、 TIEG、 PDGFC、 FGFR-1、 CGR19, et al; cell cycle modulation protein and apoptosis related factors: SMC1L1、 HMG2、 PTP4A1、 cyclin B、 CDK9、 STMN1、 melanoma antigen, et al; genes related to signaling transduction and gap junction: PRSS11、 PTP4A1、 CYR61、 GAB1、 ICAM1 (CD54)、 Integrins -β8、 connexin 43, et al. genes related to cystoskeleton, such as microtubule protein were also dominant in expression: XPO1、 GPAT、 SPP1、 LOC51174、 MIP-T3, et al. Another group of up-regulated genes were modulation factors stimulating estrogen secretion: CYP19、 IRS1 、 ADRBK2、 CRHBP, et al.
4. The microarray results showed that genes related to steroid synthesis, mitochondria synthesis, energy transportation, cellular signaling transduction, extracellular matrix formation, mucus secretion were up-regulated in the granulosa cells from mature follicles. These genes included: genes related to signaling transduction: PTPRG、 PRKCD、 KIAA010、 FDPS、 IMPA1、 TPI1、 PSPHL、 PIP5K2B, et al; genes related to extracellular matrix formation and mucus secretion: PTX3、 CALM1、 MAML1、 FN1、 Lumican、 COL5A2、 FCN1、 TIMP1, et al; genes related to mitochondria and energy supply: COX6C、 COX8、 MPP1、 MRPL9、 MRPL15、 MRPL20、 PMPCB、 TIMM8B、 ATP6J、 ATP6M、 ATP5H、 ATP6H, et al; genes related to steroid synthesis: StAR、 LOC51144、 DHCR24、 HSD11B1、 HSD17B4、 3β-HSD、 enolase 1、 SECRET, et al.
Conclusions
1. The expression difference of 462 genes between the granulosa cells from immature and mature follicles was detected by microarray. It suggests that the growth and mature of human oocytes are modulated by factors secreted by granulosa cells of follicles at different developmental stages.
2. Ten of the genes showing expression difference were confirmed by real-time RT-PCR. Except one gene showed no difference by real-time RT-PCR, the real-time RT-PCR results of other gene were consistent with results of microarray. It suggested that the results of microarray are reliable. We can further investigate the gene expression pattern and do some functional assay according to the results of microarray.
3. The expressions of 190 up-regulated genes and 270 down-regulated genes were observed in granulose cells from immatured follicles compared with matured follicles, suggesting that more regulatory factors are involved in the developmental process as the follicles grew
and matured, and the function of granulose cells of these follicles turn to be more complex.
4. We hypothesize that the roles of granulose cells at different growth stages were different. During early stage of follicular development, the granulose cells are mainly responsible for cellular proliferation, estrogen secretion and gap junction; while follicles became more mature, the granulose cells transfer to mediate progestogen secretion, signal transduction, substance transportation, extracellular matrix formation and mucus secretion.
5. The results of microarray indicated that the signaling transduction factors, growth factors, cell cycle modulation factors and paracrine factors secreted by granulosa cells are somewhat different for follicles at different stages. These factors modulate oocyte maturation, or in turn, the functions of granulosa cells are modified by the secretion from oocytes.
6. Although the results of microarray suggested us dominant gene expression during the process of follicle development, the detailed function of the genes are remained to be investigated. Further study of these genes will lead to better understanding of the mechanisms of oocyte/follicle growth and maturation.
Part III In vitro maturation—in vitro fertilization—embryo transfer—frozen-thawed of human oocytes
Objectives
To investigate the influence of ovarian follicle diameter, IVM culture media and culture duration on the number of retrieved immature oocytes, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed germinal vesicle stage and in vitro matured human oocytes. Methods
The oocytes were obtained by follicular aspiration from follicle-stimulating hormone-treated women undergoing oocyte retrieval for in vitro maturation between July 2002 and July 2004. IVM program was performed due to the possible risk of ovarian hyperstimulation in IVF-ET program. All the oocytes retrieved from follicles with 10-13.5 mm in diameter were allowed to remain in medium M-199 (TCM 199) or HTF supplemented with 10% human
plasma protein or 10% synthesized serum substance, and 0.075 IU/ml r-FSH +0.51U/mL hCG+1μg/mlβE2. Then the effects of follicle diameter, IVM culture medium and culture duration on the retrieval rate, maturation rate, fertilization rate, cleavage rate, number of high-quality embryo, implantation rate, clinical pregnancy rate, delivery rate and outcome of derived embryos transfer of in vitro matured human oocytes was investigated. Results
1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;
2. Using different culture medium (TCM 199 and HTF) had no effects on the maturation rate, fertilization rate, and cleavage rate of oocytes, however, the high-quality embryos formation rate was higher for the oocytes cultured in HTF medium (P <0.01) .
3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.
Conclusions
1. When the diameters of follicles varied and the diameter of largest oocyte exceeded 12 mm. the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased;
2. Although TCM199-based medium could improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes, the implantation rate of these embryos is still lower than the implantation rate of embryos generated in conventional IVF or ICSI cycles.
3. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy.
引文
1. Trounson A,Anderiesz C,Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51-75
2. Horie K,Takakura K,Taii S, et al.The expression of c-kit protein during oogenesis and early embryonic development.Biol Reprod,1991,45(4):547-552
3. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil,1986,76(1):31-37
4. Wynn P,Picton HM,Krapez JA.et al.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase 11 by in-vitro maruration.Hum Repro, 1998,13(11):3132-3138
5. Anderson E,Albertini DF.Gap junctions between the oocyte and companion follicle cells in the mammalian ovary.J. Cell. Biol,1976,71(2):680-686
6. Pincus G, Enzmann EV.Comparative behavior of mammalian eggs in vivo and in vitro.J Exp Med,1935,62:665-678
7. Edwards RG.Maturation in vitro of human ovarian oocytes. Lancet, 1965,2 (7419): 926-929
8. Cha KY, Koo JJ, Ko JJ, et al.Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulation cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril, 1991,55(1): 109-113
9. Trounsons A,Wood C,Kausche A.In vitro maturation and the fertilization and development competence of oocytes recovered from untreated polycystic ovarian syndrome.Fertil Steril, 1994,62(2): 353-362
10. MikkeIsen AL,Smith S,Lindenberg S.Possible factors affecting the development of oocytes in in-vitro maruration.Hum Reprod,2000,15(Suppl 5): 11-17
11. Gilchrist RB, Nayudu PL,Nowshari MA,et al.Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.Biol Reprod,1995,52(6):1234-1243
12. Mikkelsen AL, Smith S, Lindeberg S.In in-vitro maturation human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming.Hum Reprod, 1999, 14(7): 1847-1851
13. Le Du A,Kadoch IJ,Bourcigaux N,et al.In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome:the French experience.Hum Reprod,2005;20(2):420-424
14. Doson AT,Raja R,Abeyta MJ,et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet,2004,13(14):1461-1470
15. Cha KY,Han SY,Chung HM,et al.Pregnancies and delivelies after in vitro maturation culture followed by in vitro fertilization and emdryos transfer without stimulation in wemen with polycystic ovarian syndrome.Fertil Steril,2000,73(4):978-983
16. Chian RC,Buckett WM,Tulandi T,et aI.Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome.Hum Reprod,2000,15(1): 165-170
17. Chian RC,Buckett WM,Tan SL.In-vitro maturation of human oocytes.Reprod Biomed Online, 2004,8(2): 148-166
18. Child TJ,Abdui-Jalil AK,Gulekli B,et al.In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril,2001,76(5):936-942
19. Trounson A,Anderiesz C,Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51-75
20. Son WY,Lee SY,Lim JH.Fertilization,cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles.Hum Reprod,2005,20(11):3204-3207
21. Gilchrist RB,Ritter LJ,Armstrong DT.Oocyte-somatic cell interactions during follicle development in mammals.Anim Reprod Sci,2004,82-83:431-446
22. Tanghe S,Van Soom A,Nauwynck H,et al.Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization.Mol Reprod Dev,2002,61(3):414-424
23．邵华，茫烈，其木格等。去除卵丘细胞的小鼠卵母细胞在体外成熟过程中的超微结构变化。畜牧与饲料科学，2006，27(2)：17-19
24. Yuan YQ,Von SA, Leroy JLMR,et al.Apoptosis in cumulus cells,but not in oocytes, may influence bovine embryonic developmental competence.Theriogenology,2005,63(8):2147-2163
25. Hughes FM Jr, Gorospe WC.Biochemical identification of apoptosis(programmed cell death)in granulose cell: evidence for a potential mechanism underlying follicular atresia. Endocrinology, 1991,129(5): 2415-2455
26. Host E,Gabrieisen A,Lindenberg S,et al.Apoptosis in human cumulus cell in relation to zona pellucida thickness variation, maturation stage,and cleavage of the corresponding oocyte after intracystoplasmic sperm injection.Fertil Steril,2002,77(3):511-515
27．王国云，姜虹，晁岚等。人卵丘颗粒细胞的凋亡与卵子发育潜能关系的研究，生殖与避孕，2006，26(2)：91-94
28. Otsukil J, Okada A, Morimoto K,et al.The relationship between pregnancy outcome and smooth endoplasmic reticulum clusters in MII human oocytes.Human Reproduction,2004, 19(7): 1591-1597
29. Liu HC,He Z,Rosenwaks Z.Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis.Fertil Steril,2001 75(5): 947-955
30. Chin KV, Seifer DB,Feng B,et al.DNA microarray analysis of the expression profiles of luteinized granulosa cells as a function of ovarian reserve. Fertil Steril, 2002,77(6): 1214-1218
31. Mihm M,Baker PJ,Ireland JL,et al.Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle.Biol Reprod.2006.74(6): 1051-1059
32. Assoul S,Anahory T,Pantesco V,et al.The human cumulus-oocyte complex gene-expression profile.Human Reproduction,2006.21(7): 1705-1719
33. Musio A,Selicorni A,Focarelli ML,et al.X-linked Cornelia de Lange syndrome owing to SMCIL1 mutations.Nat Genet,2006;38(5):528-530
34. Wanschura S,Schoenmakers EFPM, Huysmans C,et al.Mapping of the human HMG2 gene to 4q31 .Genomics, 1996,31 (2): 264-265
35. Kajitani T,Mizutani T,Yamada K,et al.Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1,a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells.Endocrinology.2004.145(5):2307-2318
36. Zhang ZY.Mechanistic studies on protein tyrosine phosphatases.Prog Nucleic Acid Res Mol Biol,2003,73:171-220
37. Inaba T,Matsushime H,VaIentine M.et al.Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.Genomics, 1992,13(3): 565-574
38. Anderiesz C,Ferraretti AP,Magli MC,et al.Effect of recombinant human gonadotropins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum Reprod, 2000,15(5): 1140-1148
39. Liu H,Rice AP.Genomic organization and characterization of promoter function of the human CDK9 gene.Gene,2000,252(1): 51-59
40. Reynaert NL, Ckless K, Korn SH, et al. Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation. Proc Natl Acad Sci U S A. 2004, 15;101(24):8945-8950.
41. Einspanier R,Sharma HS.Scheit KH.Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells.Biochem Biophys Res Commun.1987,147(2):581-587
42. Josefsberg LB,Galiani D,Dantes A,et al.The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.Biol Reprod,2000, 62(5): 1270-1277
43. Tachibana I,Imoto M,Adjei PN,et al.Overexpression of the TGF-beta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J. Clin Invest,1997,99(10): 2365-2374.
44. Madden SL,Galella EA, Riley DB,et al.Induction of cell growth regulatory genes by p53. Cancer Res, 1996,56(23): 5384-5390
45. May Jv.The regulation of porcine theca cell proliferation in vitro:synergistic actions of ep idennal growth factor and plat-derived growth factor. Endorinology, 1992,131 (2):689-697
46. Iancu C, Mistry SJ,Arkin S,et al.Effects of stathmin inhibition on the mitotic spindle.J Cell Sci,2001,114(pt5): 909-916
47. Johnsen JI. Aurelio ON,Kwaja Z,et al.p53-mediated negative regulation of stathmin/Op18 expression is associated with G2/M cell-cycle arrest.Int J Cancer,2000,88(5): 685-691
48. Bieche I, Lachkar S,Becette V,et al.Overexpression of the stathmin gene in a subset of human breast cancer. Br J Cancer, 1998,78(6): 701-709
49. Price DK, Ball JR,Bahrani-Mostafavi Z,et al.The phosphoprotein Op18/stathmin is differentially expressed in ovarian cancer.Cancer Invest,2000,18(8):722-730
50. Guoan Chen,Hong Wang,Tarek Gharib,et al.Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas.Molecular Cellular Proteomics,2003,2(2):107-116
51. Seki N,Yoshikawa T,Hattori A,et al.cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region.J Hum Genet,2000,45(5): 309-314
52. Deiss LP. Feinstein E,Berissi H,et al.ldentification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev, 1995,9(1): 15-30
53. Gabig TG. Crean CD,Klenk A,et al.Expression and chromosomal localization of the Requiem gene.Mammalian Genome, 1998,9(8): 660-665
54. Yoshioka S, Fujiwara H,Higuchi T,et al.Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG, interleukin-1 and tumour necrosis factor-alpha.Mol Hum Reprod,2003, 9(6):311-319
55. Chien J,Staub J,Hu SI, et al.A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer.Oncogene,2004,23(8): 1636-1644
56. Bella J, Kolatkar PR, Marlor CW,et al.The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand.Proc Nat Acad Sci,1998, 95(8): 4140-4145
57. Faas MM, Moes H, van der Schaaf G,et al.Monocyte activation, but not granulocyte activation, is inhibited in the presence of developing ovarian follicles.J Reprod Immunol,2006 ,70(1-2):21-32
58. Vigano P, Gaffuri B, Ragni G,et al.Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells.J Clin Endocrinol Metab,1997,82(l):101-105
59. Hammadeh ME,Fischer-Hammadeh C,Hoffmeister H,et al.Relationship between cytokine concentrations (FGF, sICAM-1 and SCF) in serum, follicular fluid and ICSI outcome. Am J Reprod Immunol,2004,51(1):81-85
60. Babic AM, Kireeva ML, Kolesnikova TV,et al.CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Nat Acad Sci, 1998,95(11): 6355-6360
61. Martinerie C, Viegas-Pequignot E,Nguyen V,et al.Chromosomal mapping and expression of the human cyr61 gene in tumour cells from the nervous system. J Clin Path, 1997,50(6): 310-316
62. Sampath D,Zhu Y,Winneker RC,et al.Aberrant expression of Cyr61, a member of the CCN (CTGF/Cyr61/Cef10/NOVH) family, and dysregulation by 17-beta-estradiol and basic fibroblast growth factor in human uterine leiomyomas. J Clin Endocr Metab,2001,86(4): 1707-1715
63. Duncan WC,Hillier SG,Gay E,et al.Connective tissue growth factor expression in the human corpus luteum: paracrine regulation by human chrionic gonadotropin.J Clin Endocrinol Metab,2005,90(9): 5366-5376
64. Giebel J,Rune GM.Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset(Callithrix jacchus).Tissue Cell,1997,29(5):525-531
65. Teilmann SC. Differential expression and localization of connexin-37 and connexin-43 in follicles of different stages in the 4-week old mouse ovary. Mol Cell Endocrinol,2005,234(1-2):27-35
66. Wright CS,Becker DL,Lin JS.et al.Stage-specific and difierentialexpression of gap junctions in the mouse ovary:connexin-specific roles in follicular regulation.Reproduction,2001,121(1):77-88
67. Perez-Armendariz EM,Saez JC,Bravo-Moreno JF,et al.Connexion43 is expression in mouse fetal ovary .Anat Rec,2003,271(A):360-367
68. Relucenti M,Heyn R,Correr S, et al.Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.Ital J Anat Embryol,2005,110(2 Suppl 1 ):219-24
69. Salustri A,Garlanda C,Hirsch E,et al.PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.Dev,2004,131(7),1577-1586
70. Relucenti M,Heyn R,Correr S,et al.Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.Ital J Anat Embryol.2005,110(2 Suppl 1):219-224
71. Assoul S, Anahory T,Pantesco V,et al.The human cumulus-oocyte complex gene-expression profile. Human Reproduction,2006,21(7): 1705-1719
72. Zhang X, Jafari N,Barnes RB.et al.Studies of gene expression in human cumulus cells indicated pentraxin 3 as a possible marker for oocyte quality.Fertility and Sterility,2005,83 (Suppl 1): 1169-1179
73. Paffoni A, Ragni G,Doni A,et al.Follicular fluid levels of the long pentraxin PTX3.J Soc Gynecol Investig.2006, 13(3):226-231
74. Bonello N, Jasper MJ,Norman RJ.Periovulatory expression of intercellular adhesion molecule-1 in the rat ovary. Biol Reprod,2004,71(4): 1384-90
75. Masaaki I,Yasuteru M,et al.Type VI collagen expression during growth of human ovarian follicles.Fertil Stetil,2000,74(2):343-347
76. Vigano P,Fusi F,Gaffuri B,et al.Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid.Fertil Steril,1998,69(4):774-779
77. Fryer CJ, Lamar E,Turbachova I,et al.Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex.Genes Dev,2002,16( 11): 1397-1411
78. Nam Y,Weng AP,Aster JC,et al.Structural requirements for assembly of the CSL.intracellular Notch1 .Mastermind-like 1 transcriptional activation complex. J Biol Chem 2003, 278(23): 21232-21239
79. Maillard.I, Weng A P, Carpenter A, et al. Mastermind critically regulates Notch-mediated lymphoid cell fate decisions. BLOOD,2004,104(6): 1696-1702
80. Li M,Karakji EG,Xing R,et al.Expression of urokinase-type plasminogen activator and its receptor during ovarian follicular development.Endocrinology,1997,138(7):2790-2799
81. Fujii A,Harada T,Yamauchi N,et al.Interleukin-8 gene and protein expression are up-regulated by interleukin-1 beta in normal human ovarian cells and a granulosa tumor cell line. Fertil Steril, 2003, 79(1):151-157
82. Adashi EY.The potential role of interleukin-l in the ovulatory process: An evolving hypothesis. Mol Cell Endocrinol, 1998,140(1-2):77-81
83. Giles RE, Blanc H,Cann HM,et al.Matemal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA,1980,77(11):6715-6719
84. Chen X, Prosser R,Simonetti S,et al.Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet, 1995,57(2):239-247
85. Taanman JW.The mitochondrial genomes: structure, transcription, translation and replication. Biochim Biophys Acta, 1999,1410(2): 103-123
86. MCgee A, Hsuea JW.Initial and Cyclic Recruitment of Ovarian Follicles.Endocrine Reviews, 2000, 21(2): 200-214
87. Gougeon A,Testart J.Influence of human menopausal gonadotropin on the recruitment of human ovarian follicles.Fertil Steril, 1990,54(3):848-852
88. Murase M,Vemura T,Kondoh Y,et al.Role of corticotropin-releasing hormone in ovarian steroidogenesis.Endocrine,2002,18(3):255-260
89. Marion S, Robert F,Crepieux P E,et al. G protein-coupled receptor kinases and β arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells. Biol Reprod,2002,66(1):70-76
90. King SR,Ronen-Fuhramann T, Timberg R, et al.Steroid production following the in vitro transcription, translation, and mitochondrial processing of the protein products of the cDNA for the steroidogenic acute regulatory (StAR) protein.Endocrinology, 1995,136(11), 5165-5176
91. Bao B,Calder M D,Sancai Xie,et al.Expression of Steroido- genic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment,selection,and dominance of follicles of the first foiiicular wave.Bioi Reprod, 1998,59(4):953-959
92. Ronen-Fuhrmann T, Timberg R, King SRel al.Spatio-temporal expression patterns of steroidogenic acute regulatory protein(STAR)during follicular development in the rat ovary. Endocrinology, 1998, 139(1): 303-315
93. Regan L,Owen EJ,Jacobs HS.Hypersecretion of LH,infertility and miscarriage. Lancet, 1989, 336 (8724):1141-1144
94. Chian RC,Chung JT,Downey BR,Tan SL.Maturational and developmental competence of immature oocytes retrieved from bovine ovaries at different phases of folliculogenesis.Reprod Biomed Online,2002,4(2): 127-132。
95. Algriany O, Bevers M, Schoevers E,et al.Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes.Theriogenology,2004, 62(8): 1483-1497
96. El-Sheikh MM, Hussein M, Fouad S,et al.Limited ovarian stimulation (LOS), prevents the recurrence of severe forms of ovarian hyperstimulation syndrome in polycystic ovarian disease. Europ Obste Gyne Reprod Biol,2001;94(2):245-249
97．朱依敏，高惠娟，何荣环，黄荷凤。限制性促排卵方案预防卵巢过度刺激综合症的探索性研究。中华妇产科杂志，2006，41(11)：740-744
1. Abir R,Orvieto R,Dicher D, et al.Preliminary studies on apoptosis in human fetal ovaries. Fertil Steril, 2003,78(1):259-264
2. Trounson A, Anderiesz C, Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51 -75
3. Gougeon A.Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod,1986,1(2):81-87
4. Michael K.Regulation of primordial follicle assembly and development.Human Reproduction Update, 2005,11,(5): 461-471
5. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil, 1986,76(1):31-37
6. Wynn P,Picton HM,Krapez JA,et al.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation.Hum Reprod, 1998, 13(11): 3132-3138
7. Anderson E,et al.Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J.Cell.Biol, 1976,71 (2):680-686
8. Kezele P,Nilsson EE,Skinner MK.Keratinocyte growth factor acts as a mesenchymal factor that promotes ovarian primordial to primary follicle transition.Biol Reprod,2005,73(5):967-973
9. Gelety TJ,Magoffin DA.Ontogeny of steroidogenic enzyme gene expression in ovarian theca-interstitial cells in the rat:regulation by a paracrine theca differentiating factor prior to achieving luteinizing hormone responsiveness.Biol Reprod, 1997,56(4):938-945
10. Parrott JA,Skinner M K.Thecal cell granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor,hepatocyte growth factor,and Kit ligand during ovarian follicular development. Endocrinol, 1998,139 (5):2240-2245
11. Gonzalea-Bulnes A,Souza CJH,et al.Systemic and intraovarian effects of dominant follicles on ovine follicular growth.Animal Reproduction Science,2004,84(1-2):107-109
12. Schneyer AL,Fujiwara T,Fox J,et al.Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentration to circulating hormone concentrations.J Clin Endocrinol Metab,2000,85(9):3319-3330
13. Chong H,Pangas SA,Bernard DJ,et al.Structure and expression of membrane component of the inhibin receptor system.Endocrinology,2000,141(7):2600-2607
14. Seifer DB,Scott RT,Bergh PA,et al.Women with declining ovarian reserve may demonstrate a decerase in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone.J Fertil Steril, 1999,72 (1):63-65
15. Bilezikjian LM,Leal AM,Blount AL,et al.Rat anterior pituitary folliculostellate cells are targets of interleukin-beta and a major source of intrapituitary follistatin.Endocrinology,2003,144(2):732-740
16. Jesso N.Clemente N.Transduction pathway of anti-miillerian hormone,a sex-specific member of the TGF-βfamily.Trends Endocrinol Metab,2003,14(2):91-97
17. Gruijtem MJ,Visser JA,Dudinger AL,et al.Anti Milllerian hormone and its role in ovarian function.Mol Cell Endocrinol,2003,21 l(1-2):85-90
18. WeenenC,Laven JS,VonBerghAR,et al.Anti Mullerian hormone expression pattern in the human ovary:potential implications for initial and cyclic fOuicle recruitment.Mol Hum Reprod,2004,10(2):77-83
19. Weenen C,Laven JS,Von Bergh AR,et al.Anti-Mullerian hormone expression pattern in the human ovary:potential implications for initial and cyclic follicle recruitment.Molecular Human Reprod, 2004, 10(2):77-83
20. Durlinger ALL,Gruijters MJG,Kramer P,et al.Anti-mulllerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology,2002,143(3): 1076-1084
21. Durlinger ALL,Gruijters MJ,Kramer P,et al.Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.Endocrinology,2001,142(11):4891-4899
22. Visser JA,H de Jong F,Laven JSE,et al.Anti-Mullerian hormone: a new marker for ovarian function. Reproduction,2006,131 (1): 1 -9
23. Nilsson EE,Skinner MK.Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.Biology of Reproduction,2003,69(4): 1265-1272
24. Lee WS,Yoon SJ,Yoon TK,et al. Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary.Molecular and Reproductive Development, 2004 69(2): 159-163
25. Otsuka F an d Shimasaki S.A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand:Its role in regulating granulosa cell mitosis.Process ofNational Academic Science,2002,99(12):8060-8065
26. Yan C,Wang P,DeMayo J,et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Molecular Endocrinology,2001,15(6):854-866
27. Di Pasquale E,Beck-Peccoz P,Persani L.Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene.Am J Hum Genet. 2004, 75(1):106-111
28. Knight PG,Glister C.TGF-β superfamily members and ovarian follicle development. Reproduction, 2006, 132(2): 191-206
29. Hayashi M,M cgee E A,M in G,et al.Recombinant growth differentiation factor-9(GDF-9) enhance growth and diferentiation of cultured early ovarian follicle.Journal of Endocrinology, 1999, 140(3): 1236-1244
30. Vitt U A,Hsueh AJW.Stage dependent role of growth differentiation factor-9 in ovarian follicle development.Mol Cell Endocrinol,2001,183(1-2):171-177
31. Vitt U A,M azerbourg S,Klein C,et al.Bone morphogenetic protein receptor type II is a receptor for growth diferentiation factor-9.Biol Reprod,2002, 67(2):473-480
32. Albertini DF,Nishimori K,M atzuk MM.Growth differentiation factor-9 is required during early ovarian folliculogenesis.Nature, 1996,383(6600):531-535
33. Elvin JA,Yan C,W ang P,et al.Molecular characterization of the follicle defects in the growth difierentiation factor-9 deficient ovary J Mol Endocrinol, 1999,13(6):1018-1034
34. Vitt U A,M cGee E A,Hayashi M, et al. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYPI7 in ovaries of immature rats. Endocrinology, 2000, 141 (10):3814-3820
35. Hreinsson JG,Scow JE,Rasmussen C et al.Growth diferentiation factor-9 promotes the growth, development, and survival ofhum an ovarian follicles in organ culture.J Clin Endocrinol Metab, 2002, 87(1): 316-321
36. Otsuka F,Moore RK.Shimasaki S.Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary .Journal of Biological Chemistry ,2001,276(35): 32889-32895
37. Yamamoto N,Christenson LK,McAllister JM,et al.Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells.J Clin Endocrinol Metab, 2002, 87(6):2849-56
38. Yan C,W ang P,DeMayo J,et al.Synergistic role of bone morphogenetic protein 15 and growth difierentiation factor-9 in ovarian function.Mol Endocri,2001,15(6):854-866
39. Hutchinson LA,Findlay JK,de Vos FL,et al.Effects of bovine inhibin,transforming growth factor-beta and bovine activin-A on granulosa cell differentiation.Biochemical and Biophysical. Research Communications,1987,146(3):1405-1412
40. Fournet N,Weitsman SR,Zachow RJ et al.Transforming growth factor-beta inhibits ovarian 17 alpha-hydroxylase activity by a direct noncompetitive mechanism.Endocrinology, 1996,137(1): 166-174
41. Matsuyama S,Shiota K,Takahashi M.Possible role of transforming growth factor-beta as a mediator of luteotropic action of prolactin in rat luteal cell cultures.Endocrinology, 1990,127(4): 1561-1567
42. Reynaud K,Conndt R,Smiitz J,et alEffect s of kit liangd and anti-kit antibody on growt h of cultured mouse preaantral follicles.M olecular Reproduction and Development,2000,56(4):483-494
43. Parrott JA,Skinner MK.Kitligand/stem eell factor Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis.Endocrinology,1999,140(9): 4262-4271
44. Faddy M J.Folide dynamics dunng ovarian ageillg.MolCen Endocrinol,2000,163(1-2):143-148
45. Parrott JA,SkinnerMK.Kitligand actions on ovarian stroreal ceilsI effects on theca cell recruitment and steroid production.Mol Reprod Dev,2000,55(1):155-164
46. Ismail RS,Okawara Y,Fryer JN et al.Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.Mol Reprod Dev,1996,43(4):458-469
47. Otsuka F,Shimasaki S.A negative feedback system between ooeyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis.Proc Natl Acad Sci U S A,2002,99(12):8060-8065
48. Sakata S.Sakamaki K,Watanabe K,et al.Involvement of death receptor Fas in germ cell degeneration in gonads of Kit-deficient Wv/Wv mutant mice.Cell Death Differ,2003,10(6):676-686
49. Yamamoto S,KonishiI,NanbuK,et al.Immun ohistochemical localization of basic fibroblast growth factor (bFGF)during folliculogenesis in the human ovary.Gynecol Endocrinol,1997, 11(4):223-230
50. Nilsson E, Parrott J A, Skinner M K.Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis.Mol Cell Endocrinol,2001.25:175(1-2): 123-130
51. Nilsson EE,Skinner MK.Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition.Molecular and Cellular Endocrinology. 2004. 214(1-2): 19-25
52. Nilsson EE,Kezele P,Skinner MK.Leukemia inhibitory factor (LIF)promotes the primordial to primary follicle transition in rat ovaries.Molecular and cejjular Endocrinology,2002,188( 1 ):65-73
53. Baumann H,Symes A,Comeau M,et al.Multiple regions within the cytoplasm ic domains of the leukemia inhibitory factot receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells.Mol Cell Biol,1994,14(1):138-146
54. Dissen GA,Romero C,Hirshfield AN,et al.Nerve growth factor is required for early follicular development in the mammalian,Journal of Endocrnology,2001,142(5):2078-2086
55. Ozornek M H,Bielfeld P,Krussel JS,et al.Epidermal growth factor and leukemia inhibitory factor levels in follicular fluid.Association with in vitro fertilization outcome.J Reprod Med,1999,44(4):367-369
56. Dissen GA,Romero C,Hirshfield AN,et al.Nerve growth factor is required for early follicular development in the mammalian.Journal of Endocrnology,2001,142(5):2078-2086
57. Dissen RA,Robinson LL,Brooks J,et al.Neurotropins an d their receptors are expressed in the human fetal ovary.J Clin Endocrinol Metab,2002,87(2):890-897
58. Conover CA,Faessen GF,Chandrasekher YA,et al.Pregnancy-associated plasma protein-A is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa ceils and is a marker of dominant follicle selection and the corpus luteum.Endocrinology,2001,142(5):2155-2158
59. El-Roeiy A.Chen X.Roberts VJ.et al.Expression of the genes encoding the insulin-like growth factors (IGF-I and II), the IGF and insulin receptors, and IGF-binding proteins-1-6 and the localization of their gene products in normal and polycystic ovary syndrome ovaries.J Clin Endocrinol Metab, 1994, 78(6): 1488-1496
60. Baker J,Hardy MP,Zhou J,et al.Effects of an IGF I gene null mutation on mouse reproduction.Mol Endocrinol, 1996,10(5):903-918
61. Zhou J,Kumar R,Matzuk MM,et al.Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary.Mol.Endocrinol, 1997,11(13):1924-1933
62. Yuan W,Giudice LC.Insulin-like growth factor- II mediates the steroidogenic and growth proting actions of follicle stimulating hormone on human ovarian pre-antral follicles cultured in vitro. J clin Endocrinol Metab, 1999,84(4): 1479-1482
63. Spicer IJ.Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles:a control median ism for selection of dominant follicles.Biol Reprod,2004,70(51): 1223-1230
64. Hourvitz A.Kuwahara A,Hennebold JD,et al.The regulated expression of the pregnancy-associated plasma protein-a in the rodent ovary: a proposed role in the development of dominant follicles and of corpora Iutea.Endocrinoiogy,2002,143(5): 1833-1844
65. Hourivitz A,Widger AE,Filho FL.et al.Pregnancy associated plasma protein-a gene expression in human ovaries is restricted to healthy follicles and corpora lutea.J Clin Endocrinol Metab,2000,85 (12): 4916-4919
66. Westergaard LGYding Andersen,Byskov AGEpidermal growth factor in small antral ovarian follicles of pregnant women.Endocrinology, 1990,127(2):363-367
67. Das K,Stout LE,HensIeigh HC, et al.Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes.Fertil Steril, 1991,55(5):1000-1004
68. Pang Y,Ge W.Epidermal growth factor and TGF promote zebrafish oocyte maturation in vitro: Potential role of the ovarian activin regulatory system.Endocrinology,2002,143(1):47-54
69. Boiamba D,Floyd AA.McGlone JJ,et al.Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles.Boil Reprod,2002,67(1):154-160
70. Ben-Ami I,Freimann S,Armon L,et al.Novel function of ovarian growth factors: combined studies by DNA microarray, biochemical and physiological approaches.Mol Hum Reprod,2006,12(7): 413-419
71. Balasch J.Guimera M,Martinez-Pasarell O,et al.Adrenomedullin and vascular endothelial growth factor factor production by follicular fluid macrophages and granulosa eells.Hum Reprod,2004,19(4):808-814
72. Ryan NK,Woodhouse CM,Van der Hook KH,et al.Expression of leptin and its receptor in the murine ovary possible in the regulation of oocyte maturation.Biol Reprod,2002,66(11): 1548-1554
73. Hussein MR,Haemel AK,Wood GS.Apoptosis and melanoma: molecular mechanisms. J Pathol, 2003,199(3),275-288
74. Hussein HR,Haemel AK,Wood Gs.p53-related pathways and the molecular pathogenisis of melanoma.Eur J cancer Prev,2003,12(2):93-100
75. Tilly JL,Tilly KI,Kenton MI,et al.Expression of members of the bcl-2 gene family in immature ovary equiue chorionic gonadotropin mediated inhibition Of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bax long messenger rihonucleic acid levels. Endocrinology, 1995,136(1):232-247
76. Matsuda-Minehata F,GotoY,Inoue N,et al.Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcing ovaries.Mol Reprod Dev,2005,72(2): 145-151
77. Inoue J,Ishida T,Tsukamoto N,et al.Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling.Exp Cell Res,2000,10;254(1):14-24
78. Raisova M,Bektas M,Wieder T,et al.Resistance to CD95/Fas-induced and ceramide-mediated apoptosis of human melanoma cells is caused by a defective mitochondrial cytochrome c release. FEBS Lett, 2000,473(1):27-32
79. Malinin NL.Boldin MP.Kovalenko AV,et al.MAP3Krelated kinase involved in NF-kappaB induction by TNF.CD95 and IL-l.Nature.1997,385(6616):540-544
80. Franco AV,Zhang XD,Van Berkel E,et al.The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAlL)-induced apoptosis of melanoma cells.J Immunol,2001,166(9):5337-5345
81. Oltvai ZN,Millman CL,Korsraeyen SJ.Bcl-2 heterodimeraes in vivo with a conserved homolog bax thataccelerates programed cell death.Cell,1993,74(4):609-619
82. Johnson AL,Bridgham JT.Caspase-mediated apoptosis in the vertebrate ovary.Reproduction, 2002, 124(1): 19-27
83. Xiao CW,Ash K,Tsang BK.Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulose cells from tumor necrosis factor alpha-induced apoptosis. Endocrinology, 2001,142(2):557-563
84. Wang Y,Asselin E,Tsang BK.Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone.Biol Reprod,2002,66(6): 1672-1680
85. De Felici M.Regulation of primordial germ cell development in the mouse.Int J Dev Biol, 2000, 44(6):575-580
86. Giebel J,Rune GM.Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset (Callithrix jacchus).Tissue Cell,1997,29(5):525-531
87. Iwai M, Hasegawa M,Taii S,et al.Endothelins inhibit luteinization of cultured porcine granulosa cells.Endocrinology, 1991,129(4), 1909-1914
88. Schams D,Berisha B,Neuvians T,et al.Real-time changes of the local vasoactive peptide systems (angiotensin, endothelin) in the bovine corpus luteum after induced luteal regression.Mol Reprod Dev, 2003(1): 57-66
89. Huntriss J,Hinkins M,Picton HM.cDNA cloning and expression of the human NOBOX gene in oocytes and ovarian follicles.Molecular Human Reproduction,2006,12(5):283-289
90. Rajkovic A, Pangas SA, Ballow D,et al.NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science,2004,305(5687):1157-1159
91. Robles R,Morita Y,Mann KK,et al.The aryl hydrocarbon receptor, a basic helix-loophelix transcription factor of the PAS gene family,is required for normal ovarian germ cell dynamics in the mouse. Endocrinology, 2000,141 (1):450-453
92. Holly A,LaVoie,George L,et al.Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy. Molecular and Cellular Endocrinology ,2004,227,(1-2):31 -40
93. Vaskivuo TE,Anttonen M,Herva R,et al.Survival of human ovarian follicles from fetal to adult life: apoptosis,apoptosis related proteins,and transcription factor GATA-4.J Clin Endocrinol Metab, 2001, 869(7):3421-3429
94. Li J.Kim JM,Liston P,et al. Expression of inhibitor of apoptosis proteins (IAPs) in rat granulosa cells during ovarian follicular development and atresia.Endocrinology,1998, 139(3): 1321-1328
95. Komatsu K Manabe N,Kiso M,et al.SolubleFas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries.Mol Reprod Dev,2003,65(4):345-352
96. Amsterdam A,Keren-Tal I,Aharoni D,et al.Steroidogenesis and apoptosis in the mammalian ovary.Steroids,2003,(10-13) 68: 861-867
97. Clark BJ,Combs R,Hales KH,et al.Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells. Endocrinology, 1997, 138(11): 4893-4901
98. King,SR,Ronen-Fuhramann T,Timberg, R,et al.Steroid production following the in vitro transcription, translation, and mitochondrial processing of the protein products of the cDNA for the steroidogenic acute regulatory (StAR) protein.Endocrinology, 1995,136(11):5165-5176
99. Clark BJ,Wells J,King SR,et al.The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).J Biol Chem, 1994,269(45):28314-28322
100. Zhu X,Birnbaumer LG.protein subunits and the stimulation of phospholipase C by Gs-and Gicoupled receptors: Lack of receptor selectivity of Gα (16) and evidence for a synergic interaction between Gβγ and the αsubunit of a receptor activated G protein.Proc Natl Acad Sci USA,1996,93(7):2827-2831
101. Stocco DM,Clark BJ,Reinhart AJ,et al.Elements involved in the regulation of the StAR gene. Mol Ceil Endocrino,2001,177(1):55-59
102. Manna PR,Wang XJ,Stocco DM.Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression.Steroids,2003, 68(14): 1125-1134
103. Tremblay JJ,Hamel F,Viger RS.Protein kinase A-dependent cooperation between GATA and CCAAT/ enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity.Endocrinology.2002,143(10):3935-3945
104. Nishimura R,Shibaya M,Skarzynski DJ,et al. Progesterone stimulation by LH involves the phospholipase-C pathway in bovine luteal cells.J Reprod Dev,2004,50(2):257-261
105. Irusta G,Parborell F,Peluffo M,et al. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod,2003,68(5): 1577-1583
106. Manna PR,Huhtaniemi IT, Wang XJ,et al.Mechanisms of epidermal growth factor signaling: regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells.Biol Reprod,2002,67(5): 1393-1404
107. Shen WJ,Patel S.Natu V,et al.Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein : facilitation of cholesterol transfer in adrenal. J Biol Chem,2003,278(44):43870-43876
108. Hasegawa T,Liping Zhao Kathleen M.Caron,Gregor Majdic,et al.Developmental Roles of the Steroidogenic Acute Regulatory Protein (StAR) as Revealed by StAR Knockout Mice.Molecular Endocrinology, 2000,14(9): 1462-1471
109. Bao B.Calder M D,Sancai Xie,et al.Expression of Steroido- genic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment,selection,and dominance of follicles of the first foiiicular wave.Bioi Reprod, 1998,59(4):953-959
110. Ronen-Fuhrmann T,Timberg R,King S R,el al.Spatio-temporal expression patterns of steroidogenic acute regulatory protein(STAR)during foiiicular development in the rat ovary. Endocrinology, 1998, 139(1): 303-315
111. Minegishi M,Tsuchiya T, Hirakawa K,et al.Expression of steroidogenic acute regulatory protein (StAR) in rat granulosa cells.Life Sciences,2000,67(9):1015-1024
112. Srivastava VK,VijayanE,Hiney KJ,et al.Effect of ethanol on follicle stimulating hormone-induced steroidogenic acute regulatory protein (StAR) in cultured rat granulosa cells. Alcohol,2005,(37): 105-111
113. Pescador N,Houde A,Stocco D M,et al.Follicle-stimulating hormone and intraceliular second messengers regulate steroidogenic acute regulator,protein messenger ribonucleic acid in iuteinized porcine granulosa cells[J].Bioi Reprod, 1997,57(3):660-668
114. Yang P,Roy SK.A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop.Biol Reprod,2006,75(1): 149-157
115. Cannon JD,Cherian-Shaw M,Lovekamp-Swan T,et al.Granulosa cell expression of G1/S phase cyclins and cyclin-dependent kinases in PMSG-induced follicle growth.Mol Cell Endocrinol.2007,264(1-2):6-15
116. Muniz LC,Yehia G,Memin E,et al.Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells.Biol Reprod,2006,75(2):279-288
117. Liu L,Rajareddy S,Reddy P,et al.Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a.Development,2007,134(1): 199-209
118. Tomanek M,Chronowska E.Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary.Folia Histochem Cytobiol,2006,44(4):269-274
119. Yoshida N,Mita K.Yamashita M.Comparative study of the molecular mechanism of oocyte maturation in amphibians.Comp Biochem Physiol B Biochem Mol Biol,2000,126(2):189-197
120. Dekel N.Cellular,biochemical and molecular mechanisms regulating oocyte maturation.Mol and Cellular Endocrinol,2005,234(1): 19-25
121. Zhang P,Kerkela E,Skottman H,et al.Distinct sets of developmentally regulated genes that are expressed by human oocytes and human embryonic stem cells.Fertil Steril.2007.87(3):677-690
122. Gilchrist RB,Thompson JG.Oocyte maturation:Emerging concepts and technologies to improve developmental potential in vitro.Theriogenology,2007,67(1): 6-15
123. Vogelstein B,Kinzler KW.Cancer genes and the pathways they control.Nat Med,2004, 10(8):789-799
124. Hong A,Lee-Kong S,Iida T,et al.The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I.Development,2003,130(7): 1235-1242
125. Font de Mora J,Uren A,Heidaran M,et al.Biological activity of p27kipl and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes.Oncogene, 1997,15(21):2541-2551
126. Bayrak A,Oktay K.The expression of cyclin-dependent kinase inhibitors p15,p16,p21,and p27 during ovarian follicle growth initiation in the mouse.Reprod Biol Endocrinol,2003, 1:41
1
27. Einspanier R,Sharma HS,Scheit KH.Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells.Biochem Biophys Res Commun,1987,147(2):581-587
128. Josefsberg LB,Galiani D,Dantes A,et al.The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.Biol Reprod,2000,62(5): 1270-1277
129. Shao R,Rung E,Weijdegard B,Billig H.Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro.Mol Reprod Dev, 2006,73(1):50-60
130. DOWllS SM.Hudson ER,Hardie DG.A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes.Dev Biol,2002,245(1):200-212
131. MaS,LanG,MiaoY,et al.Hypoxanthine (Hx) inhibition of in vitro meiotic resumption in goat oocytes.Mol Reprod Dev,2003,66(3):306-313
132. Grimwade BG,M uskavitch MA.Artavanis-Tsakonas S,el al.The molecular genetics of the Notch locus in Drosophila melanogaster.Dev Biol.1985. 107(2):503-519
133. Kobayashi H.Shinohara H.Cotoh J.et al.Anti metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent.Br J Cancer, 1995,72(5): 1131-1137
134. Mumm JS,Kopan R.Notch signaling: from the outside in.Dev Biol,2000,228(2): 151-165
135. Artavanis-Tsakonas S,Rand MD,Lake RJ.Notch signalingxell fate control and signal integration in development.Science, 1999,284 (5415):770-776
136. Lammert E,Brown J,Melton DA.Notch gene expression during pancreatic organogenesis. Mech Dev,2000,94(1-2): 199-203
137. Milner LA,Bigas A.Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation.Blood, 1999,93(8):2431 -2448
138. Callahan R,Egan SE.Notch signaling in mammary development and oncogenesis.J Mammary Gland Biol Neoplasia.2004.9(2): 145-163
139. Karsan A.The role of notch in modeling and maintaining the vasculature.Can J Physiol Pharmacol,2005,83(1): 14-23
140. Yoon K,Gaiano N.Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci,2005,8(6):709-715
141. Johnson J,Espinoza T,McGaughey RW,et al.Notch pathway genes are expressed in mammalian ovarian follicles.Mechanisms of Development,2001,109,(2): 355-361
142. Vorontchikhina MA,Zimmermann RC,Shawber CJ,et al.Unique patterns of Notch1,Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns,2005,5, (5) :701-709
143. Fryer CJ,Lamar E.Turbachova I,et al.Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex.Genes Dev,2002,16(11): 1397-1411
144. Nam Y,Weng AP,Aster JC,et al.Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex.J.Biol.Chem,2003,278(23):21232-21239
145. Wu L,Sun T,Kobayashi K,et al.Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors.Mol Cell Bio,2002,22(21):7688-7700
146. Lin SE,Oyama T,Nagase T,Harigaya K,et al.Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling.J Biol Chem,2002,277(52): 50612-50620
147. Weng AP,Nam Y,Wolfe MS,et al.Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling.Mol Cell Biol,2003,23(2):655-664
148. Ivan Maillard,Andrew P,Weng,Andrea C.Carpenter,et al.Mastermind critically regulates Notch-mediated lymphoid cell fate decisions.BLOOD,2004,104(6): 1696-1702
149. Bork P.The modular architecture of a new family of growth regulators related to connective tissue growth factor.FEBS Lett, 1993,327(2):125-130
150. Lau LF,Nathans D.Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells.EMBOJ,1985,4(12):3145-3151
151.Menendez JA,Mehmi I,Griggs DW,et al.The angiogenic factor CYR61in breast cancer: molecular pathology and therapeutic perspectives.Endocr Relat Cancer,2003,10(2): 141-152
152. Si W,Kang Q,Luu HH,et al.CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.Mol Cell Biol, 2006, 26(8): 2955-2964
153. Chen CC,Mo FE,Lau LF.The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts.J BioJ Chem,2001,276(50):47329-47337
154. Grzeszkiewicz TM,Lindner V,Chen N,et al.The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans.Endocrinology 2002,143(4): 1441-1450
155. O'Brien TP,Lau LF. Expression of the growth factor-inducible immediate early gene eyr61 correlates with chondrogenesis during mouse embryonic development.Cell Growth Differ, 1992,3(9):645-654
156. Kireeva ML,Mo FE,Yang GP,et al.Cyr61,a product of a growth factor-inducible immediate early gene, promotes cell proliferation, migration and adhesion.Mol Cell Biol, 1996,16(4): 1326-1334
157. Gova E,Jef RB.Role of vascular endothelial growth factor in ovarian physiology and pathology.Fertil Steril,2000,74(3):429- 437
158. Berisha B,SchmnaD,Kosmann M,et al.Expressiona nd tissue concentration of vascu lar endothelialg rowth factor,,itsr eceptors, and localization in the bovine corpus luteum duringe strous cycle.Biol Reprod,2000,63:1106-1114
159. Wandji SA.Gadaby JE,Barber JA,et al.Messenger ribonucleic acid MAC25 and connective tissue growth (CTGF) are inversely gulated during folliculogenesis and early luteogensaia. Endocrinology, 2000, 141(7): 2648-2657
160. Wandji SA,Gabsby JE,Barber JR,et al.Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinol, 2000, 141(7):2648-2657
161. Duncan WC,Hillier SG,Gay E,et al.Connective tissue growth factor expression in the human corpus luteum:paracrine regulation by human chrionic gonadotropin.J Clin Endocrinol Metab,2005,90(9): 5366-5376
162. Yu J,Prado GN,Schreiber B,et al.Role of prostaglandin E(2) EP receptors and c-AMP in the expression of connective tissue growth factor.Arch Biochem Biophys,2002,404(2):302-308
163. Goodenough DA,Simon AM,Paul DL.Gap junctional intercellular communication in the mouse ovarian follicIe.Novartis Found Symp,1999,219:226-235
164. Bao XY,ReussL,Altenberg GA.Regulation of purified and reconstituted connexin 43 hemichannels by p rotein kinase C2 mediated phosphorylation of Serine 368.J B iol Chem,2004,279 (19) :20058-20066
165. De La Fuente R, Eppig JJ.Transcriptional activity of the mouse oocyte genomexompanion granulose cells modulate transcription and chromatin remodeling.Dev Biol,2001,229(1):224-236
166. Cecconi S,Ciccarelli C,Barberi M,et al.Granulosa cell-oocyte interactions.Eur J Obstet Gynecol Reprod Biol,2004,115(s1): 19-22
167. Wright CS,Becker DL,Lin JS,et al.Stage-specific and difierentialexpression of gap junctions in the mouse ovaryxonnexin-specific roles in follicular regulation.Reproduction,200l,121(1):77-88
168. Kennedy KL,Flovd AA,Clarkson AM,et al.Epidermal growth factor regu lation of connexin43 in cultured gran ulose cells from preantral rabbit follicles.Mol Reprod Dev,2003,64(1):61-69
169. Teilmann SC. Differential expression and localization of connexin-37 and connexion-43 in follicles of different stages in the 4-week old mouse ovary.Mol Cell Endocrinol,2005,234(1-2): 27-35
170. Acosta TJ,Miyamoto A.Vascular control of ovarian function: ovulation,corpus luteum formation and regression.Anim.Repro.Sci,2004,82-83:127-140
171. Perez-Armendariz EM,Saez JC,Bravo-Moreno JF,Lopez-OlmoV,et al.Connexion 43 is expression in mouse fetal ovary.Anat Rec, 2003,271(A):360-367
172. Tsai MY,Lan KC,Huang KE.Significance of mRNA levels of connexin37,connexin 43,and connexin 45 in luteinized granulosa cells of controlled hyperstimulated follicles.Fertil Steril,2003,80(6):1437-1443
173. Veitch GI.Gittens JE,Shao Q,et al.Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface.J Cell Sci,2004,117(pt 13):2699-2707
174. Simon AM,Goodenough DA,Li E,et al.Female fertility in mice lacking connexin37. Nature, 1997, 585(6616):525-529
175. Kidder GM,Mhawi AA.Gap iunctions and ovarian folliculogenesis.Reprod,2002,123(5):613-620
176. Bonello N,Jasper MJ,Norman RJ.Periovulatory expression of intercellular adhesion molecule-1 in the rat ovary.Biol Reprod,2004,71(4):1384-1390
177. Vigano P,Fusi F,Gaffuri B,et al.Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid.Fertil Steril,1998,69(4):774-779
178. Dubey AK,Cruz JR,Hartog B,et al.Expression of the integrin adhesion molecule during development of preimplantation human embryos.Fertial Steril,2001,76(1): 153-156
179. Makrigiannakis A,Coukos G,Christofidou-So!omidou M.N-cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular atresia and luteolysis?Am J Pathol. 1999, 154(5): 1391-1406
180. Sundfeldt K,Piontkewitz Y,Billig H,et al.E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation.J Reprod Fertil,2000,118(2):375-385
181. Machell NH,Farookhi R.E- and N-cadherin expression and distribution during luteinization in the rat ovary ,2003,125(6):791 -800
182. Nakhuda GS,Zimmermann RC,Bohlen P,et al.Inhibition of the vascular endothelial cell (VE)-specific adhesion molecule VE-cadherin blocks gonadotropin-dependent folliculogenesis and corpus luteum formation and angiogenesis.Endocrinology,2005,146(3): 1053-1059
183. Rabinovici J,Blankstein J,Goldman B,et al.In vitro fertilization and primary embryonic cleavage are possible in 17 alpha-hydroxylase deficiency despite extremely low intrafollicular 17 beta-estrdiol.J Clin Endocrinol metab,1989, 689(3): 693-697
184. Britt KL,Saunders PK,McPherson SJ,et al. Estrogen actions on follicle formation and early follicle development.Biol Reprod,2004,71 (5): 1712-1723
185. Skinner MK,Coffey RJ.Regulation of ovarian cell growth through the local production of transforming growth factor-alpha by theca cells.Endocrinol,1998,123(6):2 632-2638
186. Christenson LK,Stouffer RL,Strauss JF.Quantitative analysis of the hormone-induced hyperacetylation of histineH3 associated with the steroidogenlc acute regulatory protein gene promoter.J Biol Chem,2001,276 (29):392-399
187. Evans AC,Fortune JE.Selection of the dominant folhcle in cattle occurs in the absence of differences in the expression of messenger ribonucleic acid for gonadotropin receptors. Endocinol, 1997, 138(7): 2963-2971
188. Otsuka FR,Moore K,Wang X,et al.Essential role of the oocyte in estrogen amplification of follicle-stimulating hormone signaling in granulosa cells.Endocrinology,146(8):3362-3367
189. Hegele-Hartung C,Kuhnke J,Lessl M,et al.Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.Biol Reprod,1999, 61(5): 1362-1372
190. Peluso JJ.Multiplicity of Progesterone's Actions and Receptors in the Mammalian Ovary.Biol Reprod,2006,75(1):2-8
1. Pincus G, Enzmann EV.Comparative behavior of mammalian eggs in vivo and in vitro.J Exp Med,1935, 62:665-678
2. Edwards RG. Maturation in vitro of human ovarian oocytes.Lancet,1965,2(7419):926-929
3. Veek LL,Wortham JW, Witmyer J,et al.Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization.Fertil Steril,1983,39(5):594-602
4. Cha KY, Koo JJ,Ko JJ,et al.Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulation cycles,their culture in vitro and their transfer in a donor oocyte program.Fertil Steril,1991, 55(1):109-113
5. Trounsons A,Wood C,Kausche A.ln vitro maturation and the fertilization and development competence of oocytes recovered from untreated polycystic ovarian syndrome.Fertil Steril,1994,62(2):353-362
6. Gilchrist RB,Nayudu PL,Nowshari MA,Hodges JK.Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.Biol Reprod, 1995,52(6): 1234-1243
7. Wynn P,Picton HM,Krapez JA,Rutherford AJ.Balen AH,Gosden RG.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation.Hum Reprod 1998,13(11):3132-3138
8. Barnes FL,Sirard MA.Oocyte maturation.Semin Reprod Med,2000,18(2):123-131
9. Nogueira D,Cortvrindt R,Everaerdt B,et al.Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development. Reproduction,2005,130(2): 177-186
10. Yu J,Hecht NB,Schuhz RM.Expression of MSY2 in mouse oocytes and preimplantation embryos. Bio Reprod ,2001,65(4): 1260-1270
11. Mikkelsen AL.Smith S,Lindenberg S.Possible factors affecting the development of oocytes in in-vitro maturation.Hum Reprod,2000,15(Suppl 5 ):11-17
12. Mikkelsen AL, Andersson AM,Skekkebaek NE,et al.Basal concentration of oestradiol may predict the outcome of in-vitro maturationin regularly menstruating women.Hum Repro,2001,16(5):862-867
13. Mikkelsen AL,Smith S,Lindeberg S.In in-vitro maturation human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming.Hum Reprod, 1999, 14(7): 1847-1851
14. Le Du A, Kadoch IJ, Bourcigaux N,et al.In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome:the French experience.Hum Reprod,2005,20(2):420-424
15. Chian RC,Chung JT,Downey BR,Tan SL.Maturational and developmental competence of immature oocytes retrieved from bovine ovaries at different phases of folliculogenesis.Reprod Biomed Online 2002,4(1):127-132
16. Mikkelsen AL,Smith S,Lindeberg S.Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation.Human Reprod,2000,15(5): 1685-1690
17. Wynn P, Picton HM, Krapez JA.et al. Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. Hum Reprod 1998, 13(11):3132-3138
18. Cha KY,Han SY,Chung HM et al.Pregnancies and delivelies after in vitro maturation culture followed by in vitro fertilization and emdryos transfer without stimulation in women with polycystic ovarian syndrome. Fertil Steril,2000,73(4):978-983
19. Barnes FL,Kaushe A,TigliasJ et al.Production of embryos from in vitro-matured primary human oocyte. Fertil Steril, 1996,65(5): 1151-1156
20. Suikkari AM,Tulppala M,Tuuri T.Hovatta O,Barnes F.Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes.Hum Reprod,2000,15(3):747-751
21. Chian RC,Buckett WM,Tulandi T,Tan SL.Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000,15(1): 165-170
22. Chian RC, Buckett WM, Tan SL.In-vitro maturation of human oocytes.Reprod Biomed Online 2004, 8(2): 148-166
23. Child TJ,Abdul-Jalil AK,Gulekli B,Tan SL.In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome.Fertil Steril, 2001,76(4):936-942
24. Jin ZX,Ock SA,Tan Sl,Chian RC.What is the smallest size of follicle to respond to human chorionic gonadotropin injection? Fertil Steril,2005,84:S147
25. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil, 1986,76(1):31-37
26. Doson AT,Raja R,Abeyta MJ,et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet,2004,13:1461-1470
27. Trounson A,Wood C,Kausche A.In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients.Fertil Steril, 1994,62(2):353-362
28. Cha KY,Han SY,Chung HM,Choi DH,Lim JM,Lee WS,et al.Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome.Fertil Steril,2000,73(4):978-983
29. Yang SH,Son WY,Yoon SH,Ko Y,Lim JH.Correlation between in vitro maturation and expression of LH receptor in cumulus cells of the oocytes collected from PCOS patients in HCG-primed IVM cycles.Hum Reprod,2005,20(10):2097-2103
30. Marcus W.Jurema and Daniela Nogueira. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril, 2006,86, (5):1277-1291
31. Barnes FL,Crombie A,Gardner DK,Kausche A,Lacham-Kaplan O,Suikkari AM,et al.Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.Hum Reprod, 1995,10(12):3243-3247
32. Russell JB,Knezevich KM,Fabian KF,Dickson JA.Unstimulated immature oocyte retrieval:early versus midfollicular endometrial priming.Fertil Steril, 1997,67(2):616-620
33. Trounson A,Anderiesz C,Jones GM,Kausche A,Lolatgis N,Wood C.Oocyte maturation.Hum Reprod, 1998, 13(S3):52-62
34. Chian RC,Tan SL.Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod Biomed Online,2002,5(2):125-132
35. Hreinsson J,Rosenlund B,Friden B,et al.Recombinant LH is equally effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme:a randomized study[J].Hum Reprod,2003,18(10):2131-2136
36. Trounson A, Anderiesz C, Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51-75
37. Torre ML, Munari E,Albani Elena,et al.In vitro maturation of human oocytes in a follicle-mimicking three-dimensional coculture.Fertil Steril,2006,86(3):572-576
38. Son WY,Lee SY,Lim JH.Fertilization.cleavage and blastocyst development according to the maturation timing of oocytes in in-vitro maturation cycles.Hum Reprod,2005,20(11):3204-3207
39. Hwang JL,Lin YH,Tsai YL.In vitro maturation and fertilization of immature oocytes: a comprehensive study of fertilization techniques.J Assist Reprod Genet,2000,17(1):39-43
40. Soderstrom-Anttila V,Makinen S,Tuuri T,Suikkari AM.Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients.Hum Reprod,2005.20(6)1534-1540
41. Mikkelsen AL.Strategies in human in-vitro maturation and their clinical outcome.Reprod Biomed Online, 2005,10(5):593-599
42. Buckett WM,Chian RC,Barrington N,Dean K,Abdul-Jalil,Tan SL.Obstetric, neonatal and infant outcome in babies conceived by in vitro maturation (IVM): initial five-year results 1998-2003. Fertil Steril, 2004, 82(2):S133
43. Buckett WM,Chian R,Holzer H,Usher R,Tan SL.Congenital abnormalities and perinatal outcome in pregnancies following IVM, IVF, and ICSI delivered in a single center.Fertil Steril,2005,84(1):S80-81
44. Soderstrom-Anttila V, Salokorpi T, Pihlaja M,et al.Obstetric and perinatal outcome and preliminary results of development of children born after in vitro maturation of oocytes. Hum. Reprod. Jun 2006; 21(6): 1508-1513.
2. Horie K,Takakura K,Taii S, et al.The expression of c-kit protein during oogenesis and early embryonic development.Biol Reprod,1991,45(4):547-552
3. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil,1986,76(1):31-37
4. Wynn P,Picton HM,Krapez JA.et al.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase 11 by in-vitro maruration.Hum Repro, 1998,13(11):3132-3138
5. Anderson E,Albertini DF.Gap junctions between the oocyte and companion follicle cells in the mammalian ovary.J. Cell. Biol,1976,71(2):680-686
6. Pincus G, Enzmann EV.Comparative behavior of mammalian eggs in vivo and in vitro.J Exp Med,1935,62:665-678
7. Edwards RG.Maturation in vitro of human ovarian oocytes. Lancet, 1965,2 (7419): 926-929
8. Cha KY, Koo JJ, Ko JJ, et al.Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulation cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril, 1991,55(1): 109-113
9. Trounsons A,Wood C,Kausche A.In vitro maturation and the fertilization and development competence of oocytes recovered from untreated polycystic ovarian syndrome.Fertil Steril, 1994,62(2): 353-362
10. MikkeIsen AL,Smith S,Lindenberg S.Possible factors affecting the development of oocytes in in-vitro maruration.Hum Reprod,2000,15(Suppl 5): 11-17
11. Gilchrist RB, Nayudu PL,Nowshari MA,et al.Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.Biol Reprod,1995,52(6):1234-1243
12. Mikkelsen AL, Smith S, Lindeberg S.In in-vitro maturation human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming.Hum Reprod, 1999, 14(7): 1847-1851
13. Le Du A,Kadoch IJ,Bourcigaux N,et al.In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome:the French experience.Hum Reprod,2005;20(2):420-424
14. Doson AT,Raja R,Abeyta MJ,et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet,2004,13(14):1461-1470
15. Cha KY,Han SY,Chung HM,et al.Pregnancies and delivelies after in vitro maturation culture followed by in vitro fertilization and emdryos transfer without stimulation in wemen with polycystic ovarian syndrome.Fertil Steril,2000,73(4):978-983
16. Chian RC,Buckett WM,Tulandi T,et aI.Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome.Hum Reprod,2000,15(1): 165-170
17. Chian RC,Buckett WM,Tan SL.In-vitro maturation of human oocytes.Reprod Biomed Online, 2004,8(2): 148-166
18. Child TJ,Abdui-Jalil AK,Gulekli B,et al.In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril,2001,76(5):936-942
19. Trounson A,Anderiesz C,Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51-75
20. Son WY,Lee SY,Lim JH.Fertilization,cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles.Hum Reprod,2005,20(11):3204-3207
21. Gilchrist RB,Ritter LJ,Armstrong DT.Oocyte-somatic cell interactions during follicle development in mammals.Anim Reprod Sci,2004,82-83:431-446
22. Tanghe S,Van Soom A,Nauwynck H,et al.Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization.Mol Reprod Dev,2002,61(3):414-424
23．邵华，茫烈，其木格等。去除卵丘细胞的小鼠卵母细胞在体外成熟过程中的超微结构变化。畜牧与饲料科学，2006，27(2)：17-19
24. Yuan YQ,Von SA, Leroy JLMR,et al.Apoptosis in cumulus cells,but not in oocytes, may influence bovine embryonic developmental competence.Theriogenology,2005,63(8):2147-2163
25. Hughes FM Jr, Gorospe WC.Biochemical identification of apoptosis(programmed cell death)in granulose cell: evidence for a potential mechanism underlying follicular atresia. Endocrinology, 1991,129(5): 2415-2455
26. Host E,Gabrieisen A,Lindenberg S,et al.Apoptosis in human cumulus cell in relation to zona pellucida thickness variation, maturation stage,and cleavage of the corresponding oocyte after intracystoplasmic sperm injection.Fertil Steril,2002,77(3):511-515
27．王国云，姜虹，晁岚等。人卵丘颗粒细胞的凋亡与卵子发育潜能关系的研究，生殖与避孕，2006，26(2)：91-94
28. Otsukil J, Okada A, Morimoto K,et al.The relationship between pregnancy outcome and smooth endoplasmic reticulum clusters in MII human oocytes.Human Reproduction,2004, 19(7): 1591-1597
29. Liu HC,He Z,Rosenwaks Z.Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis.Fertil Steril,2001 75(5): 947-955
30. Chin KV, Seifer DB,Feng B,et al.DNA microarray analysis of the expression profiles of luteinized granulosa cells as a function of ovarian reserve. Fertil Steril, 2002,77(6): 1214-1218
31. Mihm M,Baker PJ,Ireland JL,et al.Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle.Biol Reprod.2006.74(6): 1051-1059
32. Assoul S,Anahory T,Pantesco V,et al.The human cumulus-oocyte complex gene-expression profile.Human Reproduction,2006.21(7): 1705-1719
33. Musio A,Selicorni A,Focarelli ML,et al.X-linked Cornelia de Lange syndrome owing to SMCIL1 mutations.Nat Genet,2006;38(5):528-530
34. Wanschura S,Schoenmakers EFPM, Huysmans C,et al.Mapping of the human HMG2 gene to 4q31 .Genomics, 1996,31 (2): 264-265
35. Kajitani T,Mizutani T,Yamada K,et al.Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1,a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells.Endocrinology.2004.145(5):2307-2318
36. Zhang ZY.Mechanistic studies on protein tyrosine phosphatases.Prog Nucleic Acid Res Mol Biol,2003,73:171-220
37. Inaba T,Matsushime H,VaIentine M.et al.Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.Genomics, 1992,13(3): 565-574
38. Anderiesz C,Ferraretti AP,Magli MC,et al.Effect of recombinant human gonadotropins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum Reprod, 2000,15(5): 1140-1148
39. Liu H,Rice AP.Genomic organization and characterization of promoter function of the human CDK9 gene.Gene,2000,252(1): 51-59
40. Reynaert NL, Ckless K, Korn SH, et al. Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation. Proc Natl Acad Sci U S A. 2004, 15;101(24):8945-8950.
41. Einspanier R,Sharma HS.Scheit KH.Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells.Biochem Biophys Res Commun.1987,147(2):581-587
42. Josefsberg LB,Galiani D,Dantes A,et al.The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.Biol Reprod,2000, 62(5): 1270-1277
43. Tachibana I,Imoto M,Adjei PN,et al.Overexpression of the TGF-beta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J. Clin Invest,1997,99(10): 2365-2374.
44. Madden SL,Galella EA, Riley DB,et al.Induction of cell growth regulatory genes by p53. Cancer Res, 1996,56(23): 5384-5390
45. May Jv.The regulation of porcine theca cell proliferation in vitro:synergistic actions of ep idennal growth factor and plat-derived growth factor. Endorinology, 1992,131 (2):689-697
46. Iancu C, Mistry SJ,Arkin S,et al.Effects of stathmin inhibition on the mitotic spindle.J Cell Sci,2001,114(pt5): 909-916
47. Johnsen JI. Aurelio ON,Kwaja Z,et al.p53-mediated negative regulation of stathmin/Op18 expression is associated with G2/M cell-cycle arrest.Int J Cancer,2000,88(5): 685-691
48. Bieche I, Lachkar S,Becette V,et al.Overexpression of the stathmin gene in a subset of human breast cancer. Br J Cancer, 1998,78(6): 701-709
49. Price DK, Ball JR,Bahrani-Mostafavi Z,et al.The phosphoprotein Op18/stathmin is differentially expressed in ovarian cancer.Cancer Invest,2000,18(8):722-730
50. Guoan Chen,Hong Wang,Tarek Gharib,et al.Overexpression of Oncoprotein 18 Correlates with Poor Differentiation in Lung Adenocarcinomas.Molecular Cellular Proteomics,2003,2(2):107-116
51. Seki N,Yoshikawa T,Hattori A,et al.cDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region.J Hum Genet,2000,45(5): 309-314
52. Deiss LP. Feinstein E,Berissi H,et al.ldentification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev, 1995,9(1): 15-30
53. Gabig TG. Crean CD,Klenk A,et al.Expression and chromosomal localization of the Requiem gene.Mammalian Genome, 1998,9(8): 660-665
54. Yoshioka S, Fujiwara H,Higuchi T,et al.Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG, interleukin-1 and tumour necrosis factor-alpha.Mol Hum Reprod,2003, 9(6):311-319
55. Chien J,Staub J,Hu SI, et al.A candidate tumor suppressor HtrA1 is downregulated in ovarian cancer.Oncogene,2004,23(8): 1636-1644
56. Bella J, Kolatkar PR, Marlor CW,et al.The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand.Proc Nat Acad Sci,1998, 95(8): 4140-4145
57. Faas MM, Moes H, van der Schaaf G,et al.Monocyte activation, but not granulocyte activation, is inhibited in the presence of developing ovarian follicles.J Reprod Immunol,2006 ,70(1-2):21-32
58. Vigano P, Gaffuri B, Ragni G,et al.Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells.J Clin Endocrinol Metab,1997,82(l):101-105
59. Hammadeh ME,Fischer-Hammadeh C,Hoffmeister H,et al.Relationship between cytokine concentrations (FGF, sICAM-1 and SCF) in serum, follicular fluid and ICSI outcome. Am J Reprod Immunol,2004,51(1):81-85
60. Babic AM, Kireeva ML, Kolesnikova TV,et al.CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth. Proc Nat Acad Sci, 1998,95(11): 6355-6360
61. Martinerie C, Viegas-Pequignot E,Nguyen V,et al.Chromosomal mapping and expression of the human cyr61 gene in tumour cells from the nervous system. J Clin Path, 1997,50(6): 310-316
62. Sampath D,Zhu Y,Winneker RC,et al.Aberrant expression of Cyr61, a member of the CCN (CTGF/Cyr61/Cef10/NOVH) family, and dysregulation by 17-beta-estradiol and basic fibroblast growth factor in human uterine leiomyomas. J Clin Endocr Metab,2001,86(4): 1707-1715
63. Duncan WC,Hillier SG,Gay E,et al.Connective tissue growth factor expression in the human corpus luteum: paracrine regulation by human chrionic gonadotropin.J Clin Endocrinol Metab,2005,90(9): 5366-5376
64. Giebel J,Rune GM.Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset(Callithrix jacchus).Tissue Cell,1997,29(5):525-531
65. Teilmann SC. Differential expression and localization of connexin-37 and connexin-43 in follicles of different stages in the 4-week old mouse ovary. Mol Cell Endocrinol,2005,234(1-2):27-35
66. Wright CS,Becker DL,Lin JS.et al.Stage-specific and difierentialexpression of gap junctions in the mouse ovary:connexin-specific roles in follicular regulation.Reproduction,2001,121(1):77-88
67. Perez-Armendariz EM,Saez JC,Bravo-Moreno JF,et al.Connexion43 is expression in mouse fetal ovary .Anat Rec,2003,271(A):360-367
68. Relucenti M,Heyn R,Correr S, et al.Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.Ital J Anat Embryol,2005,110(2 Suppl 1 ):219-24
69. Salustri A,Garlanda C,Hirsch E,et al.PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.Dev,2004,131(7),1577-1586
70. Relucenti M,Heyn R,Correr S,et al.Cumulus oophorus extracellular matrix in the human oocyte: a role for adhesive proteins.Ital J Anat Embryol.2005,110(2 Suppl 1):219-224
71. Assoul S, Anahory T,Pantesco V,et al.The human cumulus-oocyte complex gene-expression profile. Human Reproduction,2006,21(7): 1705-1719
72. Zhang X, Jafari N,Barnes RB.et al.Studies of gene expression in human cumulus cells indicated pentraxin 3 as a possible marker for oocyte quality.Fertility and Sterility,2005,83 (Suppl 1): 1169-1179
73. Paffoni A, Ragni G,Doni A,et al.Follicular fluid levels of the long pentraxin PTX3.J Soc Gynecol Investig.2006, 13(3):226-231
74. Bonello N, Jasper MJ,Norman RJ.Periovulatory expression of intercellular adhesion molecule-1 in the rat ovary. Biol Reprod,2004,71(4): 1384-90
75. Masaaki I,Yasuteru M,et al.Type VI collagen expression during growth of human ovarian follicles.Fertil Stetil,2000,74(2):343-347
76. Vigano P,Fusi F,Gaffuri B,et al.Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid.Fertil Steril,1998,69(4):774-779
77. Fryer CJ, Lamar E,Turbachova I,et al.Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex.Genes Dev,2002,16( 11): 1397-1411
78. Nam Y,Weng AP,Aster JC,et al.Structural requirements for assembly of the CSL.intracellular Notch1 .Mastermind-like 1 transcriptional activation complex. J Biol Chem 2003, 278(23): 21232-21239
79. Maillard.I, Weng A P, Carpenter A, et al. Mastermind critically regulates Notch-mediated lymphoid cell fate decisions. BLOOD,2004,104(6): 1696-1702
80. Li M,Karakji EG,Xing R,et al.Expression of urokinase-type plasminogen activator and its receptor during ovarian follicular development.Endocrinology,1997,138(7):2790-2799
81. Fujii A,Harada T,Yamauchi N,et al.Interleukin-8 gene and protein expression are up-regulated by interleukin-1 beta in normal human ovarian cells and a granulosa tumor cell line. Fertil Steril, 2003, 79(1):151-157
82. Adashi EY.The potential role of interleukin-l in the ovulatory process: An evolving hypothesis. Mol Cell Endocrinol, 1998,140(1-2):77-81
83. Giles RE, Blanc H,Cann HM,et al.Matemal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA,1980,77(11):6715-6719
84. Chen X, Prosser R,Simonetti S,et al.Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet, 1995,57(2):239-247
85. Taanman JW.The mitochondrial genomes: structure, transcription, translation and replication. Biochim Biophys Acta, 1999,1410(2): 103-123
86. MCgee A, Hsuea JW.Initial and Cyclic Recruitment of Ovarian Follicles.Endocrine Reviews, 2000, 21(2): 200-214
87. Gougeon A,Testart J.Influence of human menopausal gonadotropin on the recruitment of human ovarian follicles.Fertil Steril, 1990,54(3):848-852
88. Murase M,Vemura T,Kondoh Y,et al.Role of corticotropin-releasing hormone in ovarian steroidogenesis.Endocrine,2002,18(3):255-260
89. Marion S, Robert F,Crepieux P E,et al. G protein-coupled receptor kinases and β arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells. Biol Reprod,2002,66(1):70-76
90. King SR,Ronen-Fuhramann T, Timberg R, et al.Steroid production following the in vitro transcription, translation, and mitochondrial processing of the protein products of the cDNA for the steroidogenic acute regulatory (StAR) protein.Endocrinology, 1995,136(11), 5165-5176
91. Bao B,Calder M D,Sancai Xie,et al.Expression of Steroido- genic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment,selection,and dominance of follicles of the first foiiicular wave.Bioi Reprod, 1998,59(4):953-959
92. Ronen-Fuhrmann T, Timberg R, King SRel al.Spatio-temporal expression patterns of steroidogenic acute regulatory protein(STAR)during follicular development in the rat ovary. Endocrinology, 1998, 139(1): 303-315
93. Regan L,Owen EJ,Jacobs HS.Hypersecretion of LH,infertility and miscarriage. Lancet, 1989, 336 (8724):1141-1144
94. Chian RC,Chung JT,Downey BR,Tan SL.Maturational and developmental competence of immature oocytes retrieved from bovine ovaries at different phases of folliculogenesis.Reprod Biomed Online,2002,4(2): 127-132。
95. Algriany O, Bevers M, Schoevers E,et al.Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes.Theriogenology,2004, 62(8): 1483-1497
96. El-Sheikh MM, Hussein M, Fouad S,et al.Limited ovarian stimulation (LOS), prevents the recurrence of severe forms of ovarian hyperstimulation syndrome in polycystic ovarian disease. Europ Obste Gyne Reprod Biol,2001;94(2):245-249
97．朱依敏，高惠娟，何荣环，黄荷凤。限制性促排卵方案预防卵巢过度刺激综合症的探索性研究。中华妇产科杂志，2006，41(11)：740-744
1. Abir R,Orvieto R,Dicher D, et al.Preliminary studies on apoptosis in human fetal ovaries. Fertil Steril, 2003,78(1):259-264
2. Trounson A, Anderiesz C, Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51 -75
3. Gougeon A.Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod,1986,1(2):81-87
4. Michael K.Regulation of primordial follicle assembly and development.Human Reproduction Update, 2005,11,(5): 461-471
5. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil, 1986,76(1):31-37
6. Wynn P,Picton HM,Krapez JA,et al.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation.Hum Reprod, 1998, 13(11): 3132-3138
7. Anderson E,et al.Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J.Cell.Biol, 1976,71 (2):680-686
8. Kezele P,Nilsson EE,Skinner MK.Keratinocyte growth factor acts as a mesenchymal factor that promotes ovarian primordial to primary follicle transition.Biol Reprod,2005,73(5):967-973
9. Gelety TJ,Magoffin DA.Ontogeny of steroidogenic enzyme gene expression in ovarian theca-interstitial cells in the rat:regulation by a paracrine theca differentiating factor prior to achieving luteinizing hormone responsiveness.Biol Reprod, 1997,56(4):938-945
10. Parrott JA,Skinner M K.Thecal cell granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor,hepatocyte growth factor,and Kit ligand during ovarian follicular development. Endocrinol, 1998,139 (5):2240-2245
11. Gonzalea-Bulnes A,Souza CJH,et al.Systemic and intraovarian effects of dominant follicles on ovine follicular growth.Animal Reproduction Science,2004,84(1-2):107-109
12. Schneyer AL,Fujiwara T,Fox J,et al.Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development: relationship to circulating hormone concentration to circulating hormone concentrations.J Clin Endocrinol Metab,2000,85(9):3319-3330
13. Chong H,Pangas SA,Bernard DJ,et al.Structure and expression of membrane component of the inhibin receptor system.Endocrinology,2000,141(7):2600-2607
14. Seifer DB,Scott RT,Bergh PA,et al.Women with declining ovarian reserve may demonstrate a decerase in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone.J Fertil Steril, 1999,72 (1):63-65
15. Bilezikjian LM,Leal AM,Blount AL,et al.Rat anterior pituitary folliculostellate cells are targets of interleukin-beta and a major source of intrapituitary follistatin.Endocrinology,2003,144(2):732-740
16. Jesso N.Clemente N.Transduction pathway of anti-miillerian hormone,a sex-specific member of the TGF-βfamily.Trends Endocrinol Metab,2003,14(2):91-97
17. Gruijtem MJ,Visser JA,Dudinger AL,et al.Anti Milllerian hormone and its role in ovarian function.Mol Cell Endocrinol,2003,21 l(1-2):85-90
18. WeenenC,Laven JS,VonBerghAR,et al.Anti Mullerian hormone expression pattern in the human ovary:potential implications for initial and cyclic fOuicle recruitment.Mol Hum Reprod,2004,10(2):77-83
19. Weenen C,Laven JS,Von Bergh AR,et al.Anti-Mullerian hormone expression pattern in the human ovary:potential implications for initial and cyclic follicle recruitment.Molecular Human Reprod, 2004, 10(2):77-83
20. Durlinger ALL,Gruijters MJG,Kramer P,et al.Anti-mulllerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology,2002,143(3): 1076-1084
21. Durlinger ALL,Gruijters MJ,Kramer P,et al.Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.Endocrinology,2001,142(11):4891-4899
22. Visser JA,H de Jong F,Laven JSE,et al.Anti-Mullerian hormone: a new marker for ovarian function. Reproduction,2006,131 (1): 1 -9
23. Nilsson EE,Skinner MK.Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.Biology of Reproduction,2003,69(4): 1265-1272
24. Lee WS,Yoon SJ,Yoon TK,et al. Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary.Molecular and Reproductive Development, 2004 69(2): 159-163
25. Otsuka F an d Shimasaki S.A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand:Its role in regulating granulosa cell mitosis.Process ofNational Academic Science,2002,99(12):8060-8065
26. Yan C,Wang P,DeMayo J,et al.Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Molecular Endocrinology,2001,15(6):854-866
27. Di Pasquale E,Beck-Peccoz P,Persani L.Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene.Am J Hum Genet. 2004, 75(1):106-111
28. Knight PG,Glister C.TGF-β superfamily members and ovarian follicle development. Reproduction, 2006, 132(2): 191-206
29. Hayashi M,M cgee E A,M in G,et al.Recombinant growth differentiation factor-9(GDF-9) enhance growth and diferentiation of cultured early ovarian follicle.Journal of Endocrinology, 1999, 140(3): 1236-1244
30. Vitt U A,Hsueh AJW.Stage dependent role of growth differentiation factor-9 in ovarian follicle development.Mol Cell Endocrinol,2001,183(1-2):171-177
31. Vitt U A,M azerbourg S,Klein C,et al.Bone morphogenetic protein receptor type II is a receptor for growth diferentiation factor-9.Biol Reprod,2002, 67(2):473-480
32. Albertini DF,Nishimori K,M atzuk MM.Growth differentiation factor-9 is required during early ovarian folliculogenesis.Nature, 1996,383(6600):531-535
33. Elvin JA,Yan C,W ang P,et al.Molecular characterization of the follicle defects in the growth difierentiation factor-9 deficient ovary J Mol Endocrinol, 1999,13(6):1018-1034
34. Vitt U A,M cGee E A,Hayashi M, et al. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYPI7 in ovaries of immature rats. Endocrinology, 2000, 141 (10):3814-3820
35. Hreinsson JG,Scow JE,Rasmussen C et al.Growth diferentiation factor-9 promotes the growth, development, and survival ofhum an ovarian follicles in organ culture.J Clin Endocrinol Metab, 2002, 87(1): 316-321
36. Otsuka F,Moore RK.Shimasaki S.Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary .Journal of Biological Chemistry ,2001,276(35): 32889-32895
37. Yamamoto N,Christenson LK,McAllister JM,et al.Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells.J Clin Endocrinol Metab, 2002, 87(6):2849-56
38. Yan C,W ang P,DeMayo J,et al.Synergistic role of bone morphogenetic protein 15 and growth difierentiation factor-9 in ovarian function.Mol Endocri,2001,15(6):854-866
39. Hutchinson LA,Findlay JK,de Vos FL,et al.Effects of bovine inhibin,transforming growth factor-beta and bovine activin-A on granulosa cell differentiation.Biochemical and Biophysical. Research Communications,1987,146(3):1405-1412
40. Fournet N,Weitsman SR,Zachow RJ et al.Transforming growth factor-beta inhibits ovarian 17 alpha-hydroxylase activity by a direct noncompetitive mechanism.Endocrinology, 1996,137(1): 166-174
41. Matsuyama S,Shiota K,Takahashi M.Possible role of transforming growth factor-beta as a mediator of luteotropic action of prolactin in rat luteal cell cultures.Endocrinology, 1990,127(4): 1561-1567
42. Reynaud K,Conndt R,Smiitz J,et alEffect s of kit liangd and anti-kit antibody on growt h of cultured mouse preaantral follicles.M olecular Reproduction and Development,2000,56(4):483-494
43. Parrott JA,Skinner MK.Kitligand/stem eell factor Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis.Endocrinology,1999,140(9): 4262-4271
44. Faddy M J.Folide dynamics dunng ovarian ageillg.MolCen Endocrinol,2000,163(1-2):143-148
45. Parrott JA,SkinnerMK.Kitligand actions on ovarian stroreal ceilsI effects on theca cell recruitment and steroid production.Mol Reprod Dev,2000,55(1):155-164
46. Ismail RS,Okawara Y,Fryer JN et al.Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.Mol Reprod Dev,1996,43(4):458-469
47. Otsuka F,Shimasaki S.A negative feedback system between ooeyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis.Proc Natl Acad Sci U S A,2002,99(12):8060-8065
48. Sakata S.Sakamaki K,Watanabe K,et al.Involvement of death receptor Fas in germ cell degeneration in gonads of Kit-deficient Wv/Wv mutant mice.Cell Death Differ,2003,10(6):676-686
49. Yamamoto S,KonishiI,NanbuK,et al.Immun ohistochemical localization of basic fibroblast growth factor (bFGF)during folliculogenesis in the human ovary.Gynecol Endocrinol,1997, 11(4):223-230
50. Nilsson E, Parrott J A, Skinner M K.Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis.Mol Cell Endocrinol,2001.25:175(1-2): 123-130
51. Nilsson EE,Skinner MK.Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition.Molecular and Cellular Endocrinology. 2004. 214(1-2): 19-25
52. Nilsson EE,Kezele P,Skinner MK.Leukemia inhibitory factor (LIF)promotes the primordial to primary follicle transition in rat ovaries.Molecular and cejjular Endocrinology,2002,188( 1 ):65-73
53. Baumann H,Symes A,Comeau M,et al.Multiple regions within the cytoplasm ic domains of the leukemia inhibitory factot receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells.Mol Cell Biol,1994,14(1):138-146
54. Dissen GA,Romero C,Hirshfield AN,et al.Nerve growth factor is required for early follicular development in the mammalian,Journal of Endocrnology,2001,142(5):2078-2086
55. Ozornek M H,Bielfeld P,Krussel JS,et al.Epidermal growth factor and leukemia inhibitory factor levels in follicular fluid.Association with in vitro fertilization outcome.J Reprod Med,1999,44(4):367-369
56. Dissen GA,Romero C,Hirshfield AN,et al.Nerve growth factor is required for early follicular development in the mammalian.Journal of Endocrnology,2001,142(5):2078-2086
57. Dissen RA,Robinson LL,Brooks J,et al.Neurotropins an d their receptors are expressed in the human fetal ovary.J Clin Endocrinol Metab,2002,87(2):890-897
58. Conover CA,Faessen GF,Chandrasekher YA,et al.Pregnancy-associated plasma protein-A is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa ceils and is a marker of dominant follicle selection and the corpus luteum.Endocrinology,2001,142(5):2155-2158
59. El-Roeiy A.Chen X.Roberts VJ.et al.Expression of the genes encoding the insulin-like growth factors (IGF-I and II), the IGF and insulin receptors, and IGF-binding proteins-1-6 and the localization of their gene products in normal and polycystic ovary syndrome ovaries.J Clin Endocrinol Metab, 1994, 78(6): 1488-1496
60. Baker J,Hardy MP,Zhou J,et al.Effects of an IGF I gene null mutation on mouse reproduction.Mol Endocrinol, 1996,10(5):903-918
61. Zhou J,Kumar R,Matzuk MM,et al.Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary.Mol.Endocrinol, 1997,11(13):1924-1933
62. Yuan W,Giudice LC.Insulin-like growth factor- II mediates the steroidogenic and growth proting actions of follicle stimulating hormone on human ovarian pre-antral follicles cultured in vitro. J clin Endocrinol Metab, 1999,84(4): 1479-1482
63. Spicer IJ.Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles:a control median ism for selection of dominant follicles.Biol Reprod,2004,70(51): 1223-1230
64. Hourvitz A.Kuwahara A,Hennebold JD,et al.The regulated expression of the pregnancy-associated plasma protein-a in the rodent ovary: a proposed role in the development of dominant follicles and of corpora Iutea.Endocrinoiogy,2002,143(5): 1833-1844
65. Hourivitz A,Widger AE,Filho FL.et al.Pregnancy associated plasma protein-a gene expression in human ovaries is restricted to healthy follicles and corpora lutea.J Clin Endocrinol Metab,2000,85 (12): 4916-4919
66. Westergaard LGYding Andersen,Byskov AGEpidermal growth factor in small antral ovarian follicles of pregnant women.Endocrinology, 1990,127(2):363-367
67. Das K,Stout LE,HensIeigh HC, et al.Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes.Fertil Steril, 1991,55(5):1000-1004
68. Pang Y,Ge W.Epidermal growth factor and TGF promote zebrafish oocyte maturation in vitro: Potential role of the ovarian activin regulatory system.Endocrinology,2002,143(1):47-54
69. Boiamba D,Floyd AA.McGlone JJ,et al.Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles.Boil Reprod,2002,67(1):154-160
70. Ben-Ami I,Freimann S,Armon L,et al.Novel function of ovarian growth factors: combined studies by DNA microarray, biochemical and physiological approaches.Mol Hum Reprod,2006,12(7): 413-419
71. Balasch J.Guimera M,Martinez-Pasarell O,et al.Adrenomedullin and vascular endothelial growth factor factor production by follicular fluid macrophages and granulosa eells.Hum Reprod,2004,19(4):808-814
72. Ryan NK,Woodhouse CM,Van der Hook KH,et al.Expression of leptin and its receptor in the murine ovary possible in the regulation of oocyte maturation.Biol Reprod,2002,66(11): 1548-1554
73. Hussein MR,Haemel AK,Wood GS.Apoptosis and melanoma: molecular mechanisms. J Pathol, 2003,199(3),275-288
74. Hussein HR,Haemel AK,Wood Gs.p53-related pathways and the molecular pathogenisis of melanoma.Eur J cancer Prev,2003,12(2):93-100
75. Tilly JL,Tilly KI,Kenton MI,et al.Expression of members of the bcl-2 gene family in immature ovary equiue chorionic gonadotropin mediated inhibition Of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bax long messenger rihonucleic acid levels. Endocrinology, 1995,136(1):232-247
76. Matsuda-Minehata F,GotoY,Inoue N,et al.Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcing ovaries.Mol Reprod Dev,2005,72(2): 145-151
77. Inoue J,Ishida T,Tsukamoto N,et al.Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling.Exp Cell Res,2000,10;254(1):14-24
78. Raisova M,Bektas M,Wieder T,et al.Resistance to CD95/Fas-induced and ceramide-mediated apoptosis of human melanoma cells is caused by a defective mitochondrial cytochrome c release. FEBS Lett, 2000,473(1):27-32
79. Malinin NL.Boldin MP.Kovalenko AV,et al.MAP3Krelated kinase involved in NF-kappaB induction by TNF.CD95 and IL-l.Nature.1997,385(6616):540-544
80. Franco AV,Zhang XD,Van Berkel E,et al.The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAlL)-induced apoptosis of melanoma cells.J Immunol,2001,166(9):5337-5345
81. Oltvai ZN,Millman CL,Korsraeyen SJ.Bcl-2 heterodimeraes in vivo with a conserved homolog bax thataccelerates programed cell death.Cell,1993,74(4):609-619
82. Johnson AL,Bridgham JT.Caspase-mediated apoptosis in the vertebrate ovary.Reproduction, 2002, 124(1): 19-27
83. Xiao CW,Ash K,Tsang BK.Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulose cells from tumor necrosis factor alpha-induced apoptosis. Endocrinology, 2001,142(2):557-563
84. Wang Y,Asselin E,Tsang BK.Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone.Biol Reprod,2002,66(6): 1672-1680
85. De Felici M.Regulation of primordial germ cell development in the mouse.Int J Dev Biol, 2000, 44(6):575-580
86. Giebel J,Rune GM.Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset (Callithrix jacchus).Tissue Cell,1997,29(5):525-531
87. Iwai M, Hasegawa M,Taii S,et al.Endothelins inhibit luteinization of cultured porcine granulosa cells.Endocrinology, 1991,129(4), 1909-1914
88. Schams D,Berisha B,Neuvians T,et al.Real-time changes of the local vasoactive peptide systems (angiotensin, endothelin) in the bovine corpus luteum after induced luteal regression.Mol Reprod Dev, 2003(1): 57-66
89. Huntriss J,Hinkins M,Picton HM.cDNA cloning and expression of the human NOBOX gene in oocytes and ovarian follicles.Molecular Human Reproduction,2006,12(5):283-289
90. Rajkovic A, Pangas SA, Ballow D,et al.NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression.Science,2004,305(5687):1157-1159
91. Robles R,Morita Y,Mann KK,et al.The aryl hydrocarbon receptor, a basic helix-loophelix transcription factor of the PAS gene family,is required for normal ovarian germ cell dynamics in the mouse. Endocrinology, 2000,141 (1):450-453
92. Holly A,LaVoie,George L,et al.Expression of the GATA-4 and GATA-6 transcription factors in the fetal rat gonad and in the ovary during postnatal development and pregnancy. Molecular and Cellular Endocrinology ,2004,227,(1-2):31 -40
93. Vaskivuo TE,Anttonen M,Herva R,et al.Survival of human ovarian follicles from fetal to adult life: apoptosis,apoptosis related proteins,and transcription factor GATA-4.J Clin Endocrinol Metab, 2001, 869(7):3421-3429
94. Li J.Kim JM,Liston P,et al. Expression of inhibitor of apoptosis proteins (IAPs) in rat granulosa cells during ovarian follicular development and atresia.Endocrinology,1998, 139(3): 1321-1328
95. Komatsu K Manabe N,Kiso M,et al.SolubleFas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries.Mol Reprod Dev,2003,65(4):345-352
96. Amsterdam A,Keren-Tal I,Aharoni D,et al.Steroidogenesis and apoptosis in the mammalian ovary.Steroids,2003,(10-13) 68: 861-867
97. Clark BJ,Combs R,Hales KH,et al.Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells. Endocrinology, 1997, 138(11): 4893-4901
98. King,SR,Ronen-Fuhramann T,Timberg, R,et al.Steroid production following the in vitro transcription, translation, and mitochondrial processing of the protein products of the cDNA for the steroidogenic acute regulatory (StAR) protein.Endocrinology, 1995,136(11):5165-5176
99. Clark BJ,Wells J,King SR,et al.The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).J Biol Chem, 1994,269(45):28314-28322
100. Zhu X,Birnbaumer LG.protein subunits and the stimulation of phospholipase C by Gs-and Gicoupled receptors: Lack of receptor selectivity of Gα (16) and evidence for a synergic interaction between Gβγ and the αsubunit of a receptor activated G protein.Proc Natl Acad Sci USA,1996,93(7):2827-2831
101. Stocco DM,Clark BJ,Reinhart AJ,et al.Elements involved in the regulation of the StAR gene. Mol Ceil Endocrino,2001,177(1):55-59
102. Manna PR,Wang XJ,Stocco DM.Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression.Steroids,2003, 68(14): 1125-1134
103. Tremblay JJ,Hamel F,Viger RS.Protein kinase A-dependent cooperation between GATA and CCAAT/ enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity.Endocrinology.2002,143(10):3935-3945
104. Nishimura R,Shibaya M,Skarzynski DJ,et al. Progesterone stimulation by LH involves the phospholipase-C pathway in bovine luteal cells.J Reprod Dev,2004,50(2):257-261
105. Irusta G,Parborell F,Peluffo M,et al. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod,2003,68(5): 1577-1583
106. Manna PR,Huhtaniemi IT, Wang XJ,et al.Mechanisms of epidermal growth factor signaling: regulation of steroid biosynthesis and the steroidogenic acute regulatory protein in mouse Leydig tumor cells.Biol Reprod,2002,67(5): 1393-1404
107. Shen WJ,Patel S.Natu V,et al.Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein : facilitation of cholesterol transfer in adrenal. J Biol Chem,2003,278(44):43870-43876
108. Hasegawa T,Liping Zhao Kathleen M.Caron,Gregor Majdic,et al.Developmental Roles of the Steroidogenic Acute Regulatory Protein (StAR) as Revealed by StAR Knockout Mice.Molecular Endocrinology, 2000,14(9): 1462-1471
109. Bao B.Calder M D,Sancai Xie,et al.Expression of Steroido- genic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment,selection,and dominance of follicles of the first foiiicular wave.Bioi Reprod, 1998,59(4):953-959
110. Ronen-Fuhrmann T,Timberg R,King S R,el al.Spatio-temporal expression patterns of steroidogenic acute regulatory protein(STAR)during foiiicular development in the rat ovary. Endocrinology, 1998, 139(1): 303-315
111. Minegishi M,Tsuchiya T, Hirakawa K,et al.Expression of steroidogenic acute regulatory protein (StAR) in rat granulosa cells.Life Sciences,2000,67(9):1015-1024
112. Srivastava VK,VijayanE,Hiney KJ,et al.Effect of ethanol on follicle stimulating hormone-induced steroidogenic acute regulatory protein (StAR) in cultured rat granulosa cells. Alcohol,2005,(37): 105-111
113. Pescador N,Houde A,Stocco D M,et al.Follicle-stimulating hormone and intraceliular second messengers regulate steroidogenic acute regulator,protein messenger ribonucleic acid in iuteinized porcine granulosa cells[J].Bioi Reprod, 1997,57(3):660-668
114. Yang P,Roy SK.A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop.Biol Reprod,2006,75(1): 149-157
115. Cannon JD,Cherian-Shaw M,Lovekamp-Swan T,et al.Granulosa cell expression of G1/S phase cyclins and cyclin-dependent kinases in PMSG-induced follicle growth.Mol Cell Endocrinol.2007,264(1-2):6-15
116. Muniz LC,Yehia G,Memin E,et al.Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells.Biol Reprod,2006,75(2):279-288
117. Liu L,Rajareddy S,Reddy P,et al.Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a.Development,2007,134(1): 199-209
118. Tomanek M,Chronowska E.Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary.Folia Histochem Cytobiol,2006,44(4):269-274
119. Yoshida N,Mita K.Yamashita M.Comparative study of the molecular mechanism of oocyte maturation in amphibians.Comp Biochem Physiol B Biochem Mol Biol,2000,126(2):189-197
120. Dekel N.Cellular,biochemical and molecular mechanisms regulating oocyte maturation.Mol and Cellular Endocrinol,2005,234(1): 19-25
121. Zhang P,Kerkela E,Skottman H,et al.Distinct sets of developmentally regulated genes that are expressed by human oocytes and human embryonic stem cells.Fertil Steril.2007.87(3):677-690
122. Gilchrist RB,Thompson JG.Oocyte maturation:Emerging concepts and technologies to improve developmental potential in vitro.Theriogenology,2007,67(1): 6-15
123. Vogelstein B,Kinzler KW.Cancer genes and the pathways they control.Nat Med,2004, 10(8):789-799
124. Hong A,Lee-Kong S,Iida T,et al.The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I.Development,2003,130(7): 1235-1242
125. Font de Mora J,Uren A,Heidaran M,et al.Biological activity of p27kipl and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes.Oncogene, 1997,15(21):2541-2551
126. Bayrak A,Oktay K.The expression of cyclin-dependent kinase inhibitors p15,p16,p21,and p27 during ovarian follicle growth initiation in the mouse.Reprod Biol Endocrinol,2003, 1:41
1
27. Einspanier R,Sharma HS,Scheit KH.Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells.Biochem Biophys Res Commun,1987,147(2):581-587
128. Josefsberg LB,Galiani D,Dantes A,et al.The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.Biol Reprod,2000,62(5): 1270-1277
129. Shao R,Rung E,Weijdegard B,Billig H.Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro.Mol Reprod Dev, 2006,73(1):50-60
130. DOWllS SM.Hudson ER,Hardie DG.A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes.Dev Biol,2002,245(1):200-212
131. MaS,LanG,MiaoY,et al.Hypoxanthine (Hx) inhibition of in vitro meiotic resumption in goat oocytes.Mol Reprod Dev,2003,66(3):306-313
132. Grimwade BG,M uskavitch MA.Artavanis-Tsakonas S,el al.The molecular genetics of the Notch locus in Drosophila melanogaster.Dev Biol.1985. 107(2):503-519
133. Kobayashi H.Shinohara H.Cotoh J.et al.Anti metastatic therapy by urinary trypsin inhibitor in combination with an anti-cancer agent.Br J Cancer, 1995,72(5): 1131-1137
134. Mumm JS,Kopan R.Notch signaling: from the outside in.Dev Biol,2000,228(2): 151-165
135. Artavanis-Tsakonas S,Rand MD,Lake RJ.Notch signalingxell fate control and signal integration in development.Science, 1999,284 (5415):770-776
136. Lammert E,Brown J,Melton DA.Notch gene expression during pancreatic organogenesis. Mech Dev,2000,94(1-2): 199-203
137. Milner LA,Bigas A.Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation.Blood, 1999,93(8):2431 -2448
138. Callahan R,Egan SE.Notch signaling in mammary development and oncogenesis.J Mammary Gland Biol Neoplasia.2004.9(2): 145-163
139. Karsan A.The role of notch in modeling and maintaining the vasculature.Can J Physiol Pharmacol,2005,83(1): 14-23
140. Yoon K,Gaiano N.Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci,2005,8(6):709-715
141. Johnson J,Espinoza T,McGaughey RW,et al.Notch pathway genes are expressed in mammalian ovarian follicles.Mechanisms of Development,2001,109,(2): 355-361
142. Vorontchikhina MA,Zimmermann RC,Shawber CJ,et al.Unique patterns of Notch1,Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation. Gene Expression Patterns,2005,5, (5) :701-709
143. Fryer CJ,Lamar E.Turbachova I,et al.Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex.Genes Dev,2002,16(11): 1397-1411
144. Nam Y,Weng AP,Aster JC,et al.Structural requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 transcriptional activation complex.J.Biol.Chem,2003,278(23):21232-21239
145. Wu L,Sun T,Kobayashi K,et al.Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors.Mol Cell Bio,2002,22(21):7688-7700
146. Lin SE,Oyama T,Nagase T,Harigaya K,et al.Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling.J Biol Chem,2002,277(52): 50612-50620
147. Weng AP,Nam Y,Wolfe MS,et al.Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling.Mol Cell Biol,2003,23(2):655-664
148. Ivan Maillard,Andrew P,Weng,Andrea C.Carpenter,et al.Mastermind critically regulates Notch-mediated lymphoid cell fate decisions.BLOOD,2004,104(6): 1696-1702
149. Bork P.The modular architecture of a new family of growth regulators related to connective tissue growth factor.FEBS Lett, 1993,327(2):125-130
150. Lau LF,Nathans D.Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells.EMBOJ,1985,4(12):3145-3151
151.Menendez JA,Mehmi I,Griggs DW,et al.The angiogenic factor CYR61in breast cancer: molecular pathology and therapeutic perspectives.Endocr Relat Cancer,2003,10(2): 141-152
152. Si W,Kang Q,Luu HH,et al.CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.Mol Cell Biol, 2006, 26(8): 2955-2964
153. Chen CC,Mo FE,Lau LF.The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts.J BioJ Chem,2001,276(50):47329-47337
154. Grzeszkiewicz TM,Lindner V,Chen N,et al.The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans.Endocrinology 2002,143(4): 1441-1450
155. O'Brien TP,Lau LF. Expression of the growth factor-inducible immediate early gene eyr61 correlates with chondrogenesis during mouse embryonic development.Cell Growth Differ, 1992,3(9):645-654
156. Kireeva ML,Mo FE,Yang GP,et al.Cyr61,a product of a growth factor-inducible immediate early gene, promotes cell proliferation, migration and adhesion.Mol Cell Biol, 1996,16(4): 1326-1334
157. Gova E,Jef RB.Role of vascular endothelial growth factor in ovarian physiology and pathology.Fertil Steril,2000,74(3):429- 437
158. Berisha B,SchmnaD,Kosmann M,et al.Expressiona nd tissue concentration of vascu lar endothelialg rowth factor,,itsr eceptors, and localization in the bovine corpus luteum duringe strous cycle.Biol Reprod,2000,63:1106-1114
159. Wandji SA.Gadaby JE,Barber JA,et al.Messenger ribonucleic acid MAC25 and connective tissue growth (CTGF) are inversely gulated during folliculogenesis and early luteogensaia. Endocrinology, 2000, 141(7): 2648-2657
160. Wandji SA,Gabsby JE,Barber JR,et al.Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinol, 2000, 141(7):2648-2657
161. Duncan WC,Hillier SG,Gay E,et al.Connective tissue growth factor expression in the human corpus luteum:paracrine regulation by human chrionic gonadotropin.J Clin Endocrinol Metab,2005,90(9): 5366-5376
162. Yu J,Prado GN,Schreiber B,et al.Role of prostaglandin E(2) EP receptors and c-AMP in the expression of connective tissue growth factor.Arch Biochem Biophys,2002,404(2):302-308
163. Goodenough DA,Simon AM,Paul DL.Gap junctional intercellular communication in the mouse ovarian follicIe.Novartis Found Symp,1999,219:226-235
164. Bao XY,ReussL,Altenberg GA.Regulation of purified and reconstituted connexin 43 hemichannels by p rotein kinase C2 mediated phosphorylation of Serine 368.J B iol Chem,2004,279 (19) :20058-20066
165. De La Fuente R, Eppig JJ.Transcriptional activity of the mouse oocyte genomexompanion granulose cells modulate transcription and chromatin remodeling.Dev Biol,2001,229(1):224-236
166. Cecconi S,Ciccarelli C,Barberi M,et al.Granulosa cell-oocyte interactions.Eur J Obstet Gynecol Reprod Biol,2004,115(s1): 19-22
167. Wright CS,Becker DL,Lin JS,et al.Stage-specific and difierentialexpression of gap junctions in the mouse ovaryxonnexin-specific roles in follicular regulation.Reproduction,200l,121(1):77-88
168. Kennedy KL,Flovd AA,Clarkson AM,et al.Epidermal growth factor regu lation of connexin43 in cultured gran ulose cells from preantral rabbit follicles.Mol Reprod Dev,2003,64(1):61-69
169. Teilmann SC. Differential expression and localization of connexin-37 and connexion-43 in follicles of different stages in the 4-week old mouse ovary.Mol Cell Endocrinol,2005,234(1-2): 27-35
170. Acosta TJ,Miyamoto A.Vascular control of ovarian function: ovulation,corpus luteum formation and regression.Anim.Repro.Sci,2004,82-83:127-140
171. Perez-Armendariz EM,Saez JC,Bravo-Moreno JF,Lopez-OlmoV,et al.Connexion 43 is expression in mouse fetal ovary.Anat Rec, 2003,271(A):360-367
172. Tsai MY,Lan KC,Huang KE.Significance of mRNA levels of connexin37,connexin 43,and connexin 45 in luteinized granulosa cells of controlled hyperstimulated follicles.Fertil Steril,2003,80(6):1437-1443
173. Veitch GI.Gittens JE,Shao Q,et al.Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface.J Cell Sci,2004,117(pt 13):2699-2707
174. Simon AM,Goodenough DA,Li E,et al.Female fertility in mice lacking connexin37. Nature, 1997, 585(6616):525-529
175. Kidder GM,Mhawi AA.Gap iunctions and ovarian folliculogenesis.Reprod,2002,123(5):613-620
176. Bonello N,Jasper MJ,Norman RJ.Periovulatory expression of intercellular adhesion molecule-1 in the rat ovary.Biol Reprod,2004,71(4):1384-1390
177. Vigano P,Fusi F,Gaffuri B,et al.Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid.Fertil Steril,1998,69(4):774-779
178. Dubey AK,Cruz JR,Hartog B,et al.Expression of the integrin adhesion molecule during development of preimplantation human embryos.Fertial Steril,2001,76(1): 153-156
179. Makrigiannakis A,Coukos G,Christofidou-So!omidou M.N-cadherin-mediated human granulosa cell adhesion prevents apoptosis: a role in follicular atresia and luteolysis?Am J Pathol. 1999, 154(5): 1391-1406
180. Sundfeldt K,Piontkewitz Y,Billig H,et al.E-cadherin-catenin complex in the rat ovary: cell-specific expression during folliculogenesis and luteal formation.J Reprod Fertil,2000,118(2):375-385
181. Machell NH,Farookhi R.E- and N-cadherin expression and distribution during luteinization in the rat ovary ,2003,125(6):791 -800
182. Nakhuda GS,Zimmermann RC,Bohlen P,et al.Inhibition of the vascular endothelial cell (VE)-specific adhesion molecule VE-cadherin blocks gonadotropin-dependent folliculogenesis and corpus luteum formation and angiogenesis.Endocrinology,2005,146(3): 1053-1059
183. Rabinovici J,Blankstein J,Goldman B,et al.In vitro fertilization and primary embryonic cleavage are possible in 17 alpha-hydroxylase deficiency despite extremely low intrafollicular 17 beta-estrdiol.J Clin Endocrinol metab,1989, 689(3): 693-697
184. Britt KL,Saunders PK,McPherson SJ,et al. Estrogen actions on follicle formation and early follicle development.Biol Reprod,2004,71 (5): 1712-1723
185. Skinner MK,Coffey RJ.Regulation of ovarian cell growth through the local production of transforming growth factor-alpha by theca cells.Endocrinol,1998,123(6):2 632-2638
186. Christenson LK,Stouffer RL,Strauss JF.Quantitative analysis of the hormone-induced hyperacetylation of histineH3 associated with the steroidogenlc acute regulatory protein gene promoter.J Biol Chem,2001,276 (29):392-399
187. Evans AC,Fortune JE.Selection of the dominant folhcle in cattle occurs in the absence of differences in the expression of messenger ribonucleic acid for gonadotropin receptors. Endocinol, 1997, 138(7): 2963-2971
188. Otsuka FR,Moore K,Wang X,et al.Essential role of the oocyte in estrogen amplification of follicle-stimulating hormone signaling in granulosa cells.Endocrinology,146(8):3362-3367
189. Hegele-Hartung C,Kuhnke J,Lessl M,et al.Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.Biol Reprod,1999, 61(5): 1362-1372
190. Peluso JJ.Multiplicity of Progesterone's Actions and Receptors in the Mammalian Ovary.Biol Reprod,2006,75(1):2-8
1. Pincus G, Enzmann EV.Comparative behavior of mammalian eggs in vivo and in vitro.J Exp Med,1935, 62:665-678
2. Edwards RG. Maturation in vitro of human ovarian oocytes.Lancet,1965,2(7419):926-929
3. Veek LL,Wortham JW, Witmyer J,et al.Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization.Fertil Steril,1983,39(5):594-602
4. Cha KY, Koo JJ,Ko JJ,et al.Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulation cycles,their culture in vitro and their transfer in a donor oocyte program.Fertil Steril,1991, 55(1):109-113
5. Trounsons A,Wood C,Kausche A.ln vitro maturation and the fertilization and development competence of oocytes recovered from untreated polycystic ovarian syndrome.Fertil Steril,1994,62(2):353-362
6. Gilchrist RB,Nayudu PL,Nowshari MA,Hodges JK.Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations.Biol Reprod, 1995,52(6): 1234-1243
7. Wynn P,Picton HM,Krapez JA,Rutherford AJ.Balen AH,Gosden RG.Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation.Hum Reprod 1998,13(11):3132-3138
8. Barnes FL,Sirard MA.Oocyte maturation.Semin Reprod Med,2000,18(2):123-131
9. Nogueira D,Cortvrindt R,Everaerdt B,et al.Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development. Reproduction,2005,130(2): 177-186
10. Yu J,Hecht NB,Schuhz RM.Expression of MSY2 in mouse oocytes and preimplantation embryos. Bio Reprod ,2001,65(4): 1260-1270
11. Mikkelsen AL.Smith S,Lindenberg S.Possible factors affecting the development of oocytes in in-vitro maturation.Hum Reprod,2000,15(Suppl 5 ):11-17
12. Mikkelsen AL, Andersson AM,Skekkebaek NE,et al.Basal concentration of oestradiol may predict the outcome of in-vitro maturationin regularly menstruating women.Hum Repro,2001,16(5):862-867
13. Mikkelsen AL,Smith S,Lindeberg S.In in-vitro maturation human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming.Hum Reprod, 1999, 14(7): 1847-1851
14. Le Du A, Kadoch IJ, Bourcigaux N,et al.In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome:the French experience.Hum Reprod,2005,20(2):420-424
15. Chian RC,Chung JT,Downey BR,Tan SL.Maturational and developmental competence of immature oocytes retrieved from bovine ovaries at different phases of folliculogenesis.Reprod Biomed Online 2002,4(1):127-132
16. Mikkelsen AL,Smith S,Lindeberg S.Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation.Human Reprod,2000,15(5): 1685-1690
17. Wynn P, Picton HM, Krapez JA.et al. Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. Hum Reprod 1998, 13(11):3132-3138
18. Cha KY,Han SY,Chung HM et al.Pregnancies and delivelies after in vitro maturation culture followed by in vitro fertilization and emdryos transfer without stimulation in women with polycystic ovarian syndrome. Fertil Steril,2000,73(4):978-983
19. Barnes FL,Kaushe A,TigliasJ et al.Production of embryos from in vitro-matured primary human oocyte. Fertil Steril, 1996,65(5): 1151-1156
20. Suikkari AM,Tulppala M,Tuuri T.Hovatta O,Barnes F.Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes.Hum Reprod,2000,15(3):747-751
21. Chian RC,Buckett WM,Tulandi T,Tan SL.Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000,15(1): 165-170
22. Chian RC, Buckett WM, Tan SL.In-vitro maturation of human oocytes.Reprod Biomed Online 2004, 8(2): 148-166
23. Child TJ,Abdul-Jalil AK,Gulekli B,Tan SL.In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome.Fertil Steril, 2001,76(4):936-942
24. Jin ZX,Ock SA,Tan Sl,Chian RC.What is the smallest size of follicle to respond to human chorionic gonadotropin injection? Fertil Steril,2005,84:S147
25. Motlik J,Fulka J,Flechon JE.Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.J Reprod Fertil, 1986,76(1):31-37
26. Doson AT,Raja R,Abeyta MJ,et al.The unique transcriptome through day 3 of human preimplantation development.Hum Mol Genet,2004,13:1461-1470
27. Trounson A,Wood C,Kausche A.In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients.Fertil Steril, 1994,62(2):353-362
28. Cha KY,Han SY,Chung HM,Choi DH,Lim JM,Lee WS,et al.Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome.Fertil Steril,2000,73(4):978-983
29. Yang SH,Son WY,Yoon SH,Ko Y,Lim JH.Correlation between in vitro maturation and expression of LH receptor in cumulus cells of the oocytes collected from PCOS patients in HCG-primed IVM cycles.Hum Reprod,2005,20(10):2097-2103
30. Marcus W.Jurema and Daniela Nogueira. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril, 2006,86, (5):1277-1291
31. Barnes FL,Crombie A,Gardner DK,Kausche A,Lacham-Kaplan O,Suikkari AM,et al.Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.Hum Reprod, 1995,10(12):3243-3247
32. Russell JB,Knezevich KM,Fabian KF,Dickson JA.Unstimulated immature oocyte retrieval:early versus midfollicular endometrial priming.Fertil Steril, 1997,67(2):616-620
33. Trounson A,Anderiesz C,Jones GM,Kausche A,Lolatgis N,Wood C.Oocyte maturation.Hum Reprod, 1998, 13(S3):52-62
34. Chian RC,Tan SL.Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles. Reprod Biomed Online,2002,5(2):125-132
35. Hreinsson J,Rosenlund B,Friden B,et al.Recombinant LH is equally effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme:a randomized study[J].Hum Reprod,2003,18(10):2131-2136
36. Trounson A, Anderiesz C, Jones G.Maturation of human oocytes in vitro and their development competence.Reproduction,2001,121(1):51-75
37. Torre ML, Munari E,Albani Elena,et al.In vitro maturation of human oocytes in a follicle-mimicking three-dimensional coculture.Fertil Steril,2006,86(3):572-576
38. Son WY,Lee SY,Lim JH.Fertilization.cleavage and blastocyst development according to the maturation timing of oocytes in in-vitro maturation cycles.Hum Reprod,2005,20(11):3204-3207
39. Hwang JL,Lin YH,Tsai YL.In vitro maturation and fertilization of immature oocytes: a comprehensive study of fertilization techniques.J Assist Reprod Genet,2000,17(1):39-43
40. Soderstrom-Anttila V,Makinen S,Tuuri T,Suikkari AM.Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients.Hum Reprod,2005.20(6)1534-1540
41. Mikkelsen AL.Strategies in human in-vitro maturation and their clinical outcome.Reprod Biomed Online, 2005,10(5):593-599
42. Buckett WM,Chian RC,Barrington N,Dean K,Abdul-Jalil,Tan SL.Obstetric, neonatal and infant outcome in babies conceived by in vitro maturation (IVM): initial five-year results 1998-2003. Fertil Steril, 2004, 82(2):S133
43. Buckett WM,Chian R,Holzer H,Usher R,Tan SL.Congenital abnormalities and perinatal outcome in pregnancies following IVM, IVF, and ICSI delivered in a single center.Fertil Steril,2005,84(1):S80-81
44. Soderstrom-Anttila V, Salokorpi T, Pihlaja M,et al.Obstetric and perinatal outcome and preliminary results of development of children born after in vitro maturation of oocytes. Hum. Reprod. Jun 2006; 21(6): 1508-1513.