【作者】 朱依敏；

【导师】 黄荷凤；

【作者基本信息】 浙江大学 ， 妇产科， 2007， 博士

【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、E₂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。更多还原

【Abstract】 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 stagesObjectivesTo 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 lightmicroscopy. 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. ResultsThe 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 apparatusdecreased; 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. Conclusions1. 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 folliclesObjectivesTo 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.MethodsThe 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. Results1. 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.Conclusions1. 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 grewand 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 oocytesObjectivesTo 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. MethodsThe 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% humanplasma 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. Results1. 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.Conclusions1. 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.更多还原