【作者】 金海霞；

【导师】 孙莹璞；

【作者基本信息】 郑州大学 ， 妇产科学， 2004， 硕士

【摘要】 出生时，卵母细胞处于第一次减数分裂前期，人卵巢内约有100多万不成熟卵母细胞，青春期后，在内源性促性腺激素作用下，不成熟卵母细胞成熟并排出，人的一生中仅有400多个卵子成熟并排出，绝大多数卵母细胞在发育中逐步退化闭锁。募集那些退化的卵子将其在体外培养成熟(In vitro maturation，IVM)可提供有关卵泡发生及卵母细胞成熟的信息，并为不孕妇女提供卵子，可以节省体外受精-胚胎移植(Invitro fertilization-embryo transfer，IVF-ET)中用于募集卵泡的药物费用，减少因这些药物引起的卵巢过度刺激、体重增加、恶心、情绪不稳及可能的远期并发症，同时使病人复诊次数减少，治疗周期缩短。然而卵母细胞虽能在体外成熟、受精、胚胎移植后妊娠成功，但体外成熟率、受精率及妊娠率低。培养介质会影响甚至改变哺乳动物卵母细胞减数分裂的调节。自20世纪90年代以来，为了提高不成熟卵母细胞的体外成熟率和质量，大量的学者研究通过向培养液中添加各种物质来改善培养微环境以促进未成熟卵母细胞的体外成熟。目前，各实验室所用的体外培养系统的成分各异，至今无统一标准，寻求最佳培养系统，提高体外成熟卵母细胞的质量及成熟率至关重要。 伴随IVF-ET等人类助孕技术的发展，低温保存技术起到了不可估量的作用。卵母细胞的冷冻保存可以避免胚胎冻存所涉及的法律、伦理、道德以及宗教等因素；为那些因病变需要切除卵巢的年轻未婚未育患者保存生育力提供最大的希望；冷冻未成熟卵母细胞可与体外培养技术结合，建立“未成熟卵母细胞库”，为少卵、无卵、卵巢早衰等妇女提供卵子；为科研提供足够数量的卵母细胞，并将进一步推动生物学、遗传学、组织学等方面的发展。1977年，Whittingham首次冻存小鼠体内成熟卵母细郑州人学2仪妈涌硕上毕业论文胞获得成功，揭开了卵母细胞冷冻的序幕。1986年Chen首次冻存人卵母细胞成功并获得妊娠26周胎儿，随后对卵母细胞冻存进行了大量的研究，但整个卵子冻存技术进展缓慢，迄今为止，卵母细胞冻存成功率低，冻存方法至今无统一标准，冻融的各个坏节均可影响卵子的正常结构和功能，导致随后的存活率、受精率及胚胎发育率低。影响卵母细胞冻存的因素很多，其中有卵母细胞发育阶段及冷冻方法。成熟卵母细胞处于减数分裂的 MH期，第一极体己经排出，染色体排列在减数分裂纺锤体的赤道板上，纺锤体微管的正常排列对染色体正确排列和分离是十分重要的，冷冻解冻过程中，纺锤体易被细胞内形成的冰晶损伤，引起染色体异常，导致受精和发育失败。生殖泡期(Germinal vesicl早，GV)期卵母细胞处于减数分裂的双线期，尚未形成纺锤体，出现染色体异常的可能性小，冻存未成熟卵母细胞似乎是更好的选择。目前国外经不同冷冻方法超低温冷冻保存人类成熟或不成熟卵母细胞产生的婴儿己有出生，而国内卵母细胞的冷冻保存还处于起步阶段。尚未见有关冻存小鼠及人GV期卵母细胞及对其后体外成熟、受精和胚胎发育影响的系统研究报道。探讨影响卵母细胞冷冻的因素，寻求最佳冷冻时期和冷冻方法尤为重要。 本研究以小鼠为研究对象，一方面系统研究表皮生长因子(EPidermal gro叭hfacto几EGF)、1 7p一雌二醇(1 7beta一estradiol，1 7p一E:)、丙酮酸及保留卵丘细胞对小鼠GV期卵母细胞体外成熟、受精及随后早期胚胎发育的影响，以期优化IVM培养基，为临床提高人类卵母细胞成熟及受精后胚胎发育提供新的理论依据。另一方面探讨常规慢速冷却/快速复温及玻璃化法分别对GV及第二次减数分裂中期(MetaPhasell，MH)的卵母细胞复苏、体外成熟、受精及早期胚胎发育的影响，旨在为卵母细胞的冷冻保存寻找最佳的时期和最佳的冷冻保存方法，为其更好的应用于人类提供新的理论依据。郑州大学加04届硕}毕业沦文第一部分表皮生长因子、17p雌二醇、丙酮酸及卵丘细胞对小鼠卵母细胞体外成熟的影响 目的探讨培养液中添加表皮生长因子(EGF)、1 7p一雌二醇(1 7p·EZ)、丙酮酸及保留卵丘细胞对小鼠生殖泡期(GV)卵母细胞体外成熟、受精及其胚胎发育的影口向。 方法GV期卵母细胞来自8一12周、体重20一259的昆明雌性小白鼠。将收集到的形态较好的GV期卵母细胞随机分为5组:对照组:TCM199+lO%胎牛血清+7 SIU几FsH+o，sluzm一heG+o.osmg/ml青霉素+o.o75mg/ml链霉素;EGF组:对照组液+l ong/m IEGF;1 7p一E:组:对照组+l，g/ml 1 7p一EZ;丙酮酸组:对照液十0.3mM丙酮酸;EGF+17日一EZ+丙酮酸组:对照组液+1 ong/ml EGF+1 pg/ml 17p一EZ+o.3mM丙酮酸。五组体外成熟液中，每组又分为卵丘细胞复合物(COC)及裸卵(DO)组。培养24小时后，对已排出第一极体的卵母细胞行卵胞浆内单精子显微注射(ICSI)，注射后4一5小时，观察卵母细胞受精情况，受精后继续培养5一6天，观察成熟率、受精率、卵裂率、2细胞、8细胞、桑堪胚及囊胚形成情况。 结果 1 EGF、17p一EZ、丙酮酸对DO组未成熟卵母细胞体外成熟的影响与对照组相比，EGF组、EGF十17p·EZ+丙酮酸组DO的Mn率明显高于对照组DO，尸<0.05，生殖泡破裂(GVBD)率、受精率、卵裂率、2细胞及8细胞?更多还原

【Abstract】 There are one million immature oocytes at prophase of meiosis in ovary when women were bom. After adolescence, a number of them start to mature and ovulate under the effect of gonadotrophin. A health fertile woman will only ovalate approximately 400 of these unique cells. Oocyte resting in the ovary will die by atresia. Harvesting oocytes atresia and maturing them in vitro will help us to collect the massage of folliculogenesis and oocytes in vitro maturation (IVM), to provide oocytes for infertile women, to reduce in cost that need to purchace meducation for ovarian stimulation in IVF-ET and avoid the side effects associated with the use of gonadotrophin such as ovarian hyperstimualtion syndrome, weight gain, abdominal bloating and the long-term risk of it. At the same time, IVM can simptificate of treatment. Although, oocyte can mature, fertilization in vitro and pregenancy can succeed after embryo transfer, the rates of maturation, fertilize and pregenancy are very low. The compositon in culture medium can effect and even change the regulation of oocyte meiosis in mammal. Since 1990’s, many scientists improve maturation of immature oocyte by adding different substances to culture medium. Today, the composition of culture system in vitro is different, there is no unified standard. To find the optimum culture medium system, improve the quality and maturation of oocyte is very籋 A 1*2004 MMimportant.Following the development of IVF-ET, cryopreservation has an inestimable effects. Cryopreservation of oocyte can avoid some of the law, ethical, moral and religion problems encountered by embryo crypreservation. It can provide the best hope of preserving the eugenesis for these young women at risk of losing their ovarian function because of disesses. Immature oocyte cryopreservation combining with oocyte maturation in vitro will built an egg banking, which can provide eggs to women who have little eggs, no eggs and decline of ovarian function, provide enough eggs to research and promote the development of biology, genetics and histology. Whittingham achieved the first success of mouse oocyte cryopreservation in 1997. The first human live fetal at 26 weeks from frozen was reported by Chen in 1986. Since then, many researchers studied the oocyte cryopreservation, but the improvement of it was very slow. Today, the success of oocyte cryopreservation is very low. There is no unified standard about cryopreservation methods. Every step of freezing-thawing can effect the nomal structure and function of oocyte, causing the low survival rate, fertilization rate and poor embryo development competence. There are many factors that effect oocyte cryopreservatin. One is the stage of oocyte, the other is the methods of cryopreservation . Mature oocyte is in metaphage II stage of the meiotic division, it shows the first body extruded and the chromosomes arrange on the meiotic spindle. It is well know that appropriate organization of spindle microtubules is essential for correct alignment and segregation of chromosomes. During the freezing and thawing, spindle is more easy to be injured by intracellular ice formation, leading to chromosomal abnormity and failure of fertilization and even development. Oocyte at germinal vesicle(GV) stage is in diplotene stage of the meiotic division. The spindle has not been formed and chromosomal abnormality is very low at this stage. It is likely that cryopreservation of GV stage oocyte is a better section than MII stage oocyte. Recently, a!few fetuses from mature and immature oocyte cryopreservation by different methods have born in overseas. It is very important to explore the factors which effect oocyte cryopreservation and find the optimal stage and method.We used mouse as the objects, studied the effects of epidermal growth factor(EGF),17beta-estradiol(17p-E2), pyruvic acid and cumulus cells on developmentcompetence of in vitro maturation mouse oocyte. The purpose was to optimize the culture system of IVM and provide the new theoretics of improving the maturation, fertilization and更多还原