不同培养介质及冷冻方法对小鼠卵母细胞体外成熟及胚胎发育的影响
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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h
facto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，M
H)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?
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 ca
n 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 MM
important.
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 development
competence 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
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h
facto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，M
H)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?
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 ca
n 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 MM
important.
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 development
competence 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
引文
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[2] Whitacre KS, Seifer DB, Friedman CI, et al. Effects of ovarian source, patient age, and menstrual cycle phase on in vitro maturation of immature human ooeytes. Fertil Steril, 1998, 70(6): 1015-1021
[3] Gilula NB, Epstein ML, Beers WH. Cell-to-cell communication and ovulation: a study of the cumulus cell-oocyte complex. J Cell Biol, 1978, 78(1):58-75
[4] Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol, 1988, 125(1):181-186
[5] Fujiwara T, Nakada K, Shirakawa H, et al. Development of inositol trisphosphate-induced calcium release mechanisms during maturation of hamster oocytes. Dev Biol, 1993, 156(1):69-79
[6] Carroll J, Jones KT, Whittingham DG.. Ca~(2+) release and the development of Ca~(2+) release mechanisms during oocyte maturation. Rev Reprod, 1996, 1 (3): 137-143
[7] Pincus G, Enzmann EV. The comparative behavior of mammalian eggs in vivo and in vitro. The activation of ovarian eggs. J Exp Med, 1935, 62:665
[8] Edwards RG, Wales DS, Edin PH. Maturation in vitro of human ovarian oocytes. Lancet, 1965, 2(7419):926-929
[9] Whittingham DG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at --196 degrees C.J Reprod Fertil. 1977,49(1):89-94
[10] Chen C. Pregnancy after human oocyte cryopreservation. Lancet. 1986, 19;1(8486):884-6
[11] Porcu E, Fabbri R, Seracchioli R, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril. 1997,68(4):724-6
[12] Ledda S, Leoni G, Bogliolo L, et al. Oocyte cryopreservation and ovarian tissue
banking. Theriogenology. 2001, 1 ;55(6): 1359-71
[13] Shaw JM, Oranratnachai A, Trounson AO. Fundamental cryobiology of mammalian oocytes and ovarian tissue. Theriogenology. 2000, 1 ;53(1):59-72
[14] Hong SW, Chung HM, Lim JM, et al. Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril. 1999, 72(1):142-6
[15] Ledda S, Leoni G, Bogliolo L, et al. Oocyte cryopreservation and ovarian tissue banking. Theriogenology. 2001, 55(6): 1359-71
[16] Rail WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature. 1985,313(6003):573-5
[17] Das K, Stout LE, Hensleigh 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
[18] Rieger D, Luciano AM, Modina S, et al. The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro. J Reprod Fertil, 1998, 112(1):123-130
[19] Grupen CG, Nagashima H and Nottle MB. Role of epidermal growth factor and insulin-like growth factor-Ⅰ on porcine oocyte maturation and embryonic development in vitro. Reprod Fertil Dev, 1997, 9(6):571-575
[20] Merriman JA, Whitingham DG, Carroll J. The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes. Hum Reprod, 1998, 13(3):690-695
[21] Zelinski-wooten MB, Hess DL, Wolf DP, et al. Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fertil Steril, 1994,61(6):1147-1155
[22] Tesarik J, Mendoza C. Nongenomic effects of 17beta-estradiol on maturing human oocytes:relationship to oocyte developmental potential. J Clin Endocrinol Metab, 1995,80(4): 1438-1443
[23] Lin YW, Schuetz AW. In vitro estrogen modulatin of pituitary and
progesterone-induced oocyte maturation in Rana pipients. J Exp Zool, 1983, 226:281-291
[24] Legge M. Oocyte and zygote zona pellucida permeability to macromolecules. J Exp Zool, 1995, 271(2):145-50
[25] Wright CS, Hovatta O, Margara R, et al. Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum Reprod, 1999,14(6): 1555-62
[26] Goud PT, Goud AP, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod, 1998,13(6):1638-1644
[27] 艾继辉，罗丽兰，刘建新．表皮生长因子卵丘细胞对卵母细胞体外成熟的影响．中国实用妇科与产科杂志，2003，19(4)：209-211
[28] 吕述彦，程去英．表皮生长因子调节小鼠生发泡期卵母细胞发育成熟的实验研究．生殖医学杂志，2002，11(1)：25-30
[29] Lorenzo PL, Illera M J, Illera JC, et al. Enhancement of cumulus expansion and nuclear maturation during bovine oocyte maturation in vitro by the addition of epidermal growth factor and insulin-like growth factor I. J Reprod Fertil, 1994, 101(3):697-701
[30] Barnes FL, Sirard MA. Oocyte maturation. Semin Reprod Med, 2000,18(2):123-31
[31] De La Fuente R, O'Brien M J, Eppig JJ. Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes. Hum Reprod., 1999,14(12):3060-8
[32] Mcnatty KP. Follicular fluid. In :Jones, R.E.(Ed.), The Vertebrate Ovary. :lenum Press. New York, 1978:215-259
[33] Ainsworth L, Tsang BK, Downey BR. Interrelationship between follicular fluid steroid levels, gonadotropic stimuli , and oocyte maturation during preovulatory development of porcine follicles. Biol.Reprod, 1980,23(3):621-627
[34] Mingoti GZ, Garcia JM, Rosa-e-Silva AA. The effect of serum on in vitro maturation, fertilization and steroidogenesis of bovine oocytes co-cultured with
granulose cells. Braz J Med Biol Res, 1995, 28(2) :213-217
[35] Moudgal NR, Sherry G, Selvaraj N, et al. Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development. J Reprod Fertil suppl. 1996, 50:69-81
[36] Zheng P, Si W, Bavister B, et al. 17 β-estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys. Hum Reprod, 2003,18(10):2137-2144
[37] Dode MA, Graves, CN. Role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes. Anim Reprod Sci, 2003,78(1-2):99-110
[38] Fukui Y, Fukushima Y, Terawaki Y, et al. Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro. Theriogenology, 1982,18:161-175
[39] Younis AI, Brackett BG, Fayrer-Hosken RA. Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 1989, 23(2):189-201
[40] McGaughey RW. The culture of pig oocytes in minimal medium, and the influence of progesterone and estradiol-17beta on meiotic maturation. Endocrinology 1977,100(1):39-45
[41] Ochi T. Induction of multiple microtubule-organizing centers, multipolar spindles and multipolar division in cultured V79 cells exposed to diethylstilbestrol, estradiol- 17beta and bisphenol A. Murat Res, 1999, 431 (1): 105-121
[42] Fagbohun CF, Downs SM. Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes. J Reprod Fertil, 1992, 96(2):681-97
[43] Rieger D, Loskutoff NM. Changes in the metabolism of glucose, pyruvate, glutamine and glycine during maturation of cattle oocytes in vitro. J Reprod Fertil, 1994, 100(1):257-62
[44] Downs SM, Utecht AM. Metabolism of radiolabeled glucose by mouse oocytes and
oocyte-cumulus cell complexes. Biol Reprod, 1999,60(6): 1446-52
[45] Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil, 1984, 72(1):9-13
[46] Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci U S A, 1967,58(2):560-7
[47] Donahue RP, Stern S. Follicular cell support of oocyte maturation: production of pyruvate in vitro. J Reprod Fertil, 1968,17(2):395-8
[48] Leese H J, Barton AM. Production of pyruvate by isolated mouse cumulus cells. J Exp Zool,
[49] Downs SM, Mastropolo AM. The participation of energy substrates in the control of meiotic maturation in murine oocytes. Dev Biol, 1994,162(1): 154-68
[50] Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev Biol, 1995, 167(2):502-12
[51] Downs SM, Hudson ER, Hardie DG. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev Biol. 2002,245(1):200-12
[52] Leese HJ. Physiology of the fallopian tube: the provision of nutrients for oocytes and early embryos. In Ovulation to Implantation PP121-125 Eds JHL Evers and MJ Heineman. 1990, Elsevier Science Publishers, New York.
[53] Downs SM, Hudson ED. Energy substrates and the completion of spontaneous meiotic maturation. Zygote, 2000,8(4):339-51
[54] Geshi M, Takenouchi N, Yamauchi N, et al. Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod, 2000, 63(6): 1730-4
[55] Antczak M, Van Blerkom J. Oocyte influences on early evelopment: the regulatory proteins leptin and STAT3 are polarzed in mouse and human oocytes and differentally distributed within the cells of the preimplantation embryo. Mol Hum Reprod, 1997, 3(1):1067-86
[56] 卜淑敏，夏国良，谢辉蓉等．卵丘细胞产生的一氧化氮促进小鼠卵母细胞的成
熟．科学通报，2002，47(22)：1730-1733
[57] Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol, 1988,125(1):181-6
[58] Yoshida M. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol Reprod Dev, 1993,35(1):76-81
[59] Eppig J J, Downs SM. Chemical signals that regulate mammalian oocyte maturation. Biol Reprod, 1996, 54:197-207
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[15] Ledda S, Leoni G, Bogliolo L, et al. Oocyte cryopreservation and ovarian tissue banking. Theriogenology. 2001, 55(6): 1359-71
[16] Rail WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature. 1985,313(6003):573-5
[17] Das K, Stout LE, Hensleigh 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
[18] Rieger D, Luciano AM, Modina S, et al. The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro. J Reprod Fertil, 1998, 112(1):123-130
[19] Grupen CG, Nagashima H and Nottle MB. Role of epidermal growth factor and insulin-like growth factor-Ⅰ on porcine oocyte maturation and embryonic development in vitro. Reprod Fertil Dev, 1997, 9(6):571-575
[20] Merriman JA, Whitingham DG, Carroll J. The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes. Hum Reprod, 1998, 13(3):690-695
[21] Zelinski-wooten MB, Hess DL, Wolf DP, et al. Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fertil Steril, 1994,61(6):1147-1155
[22] Tesarik J, Mendoza C. Nongenomic effects of 17beta-estradiol on maturing human oocytes:relationship to oocyte developmental potential. J Clin Endocrinol Metab, 1995,80(4): 1438-1443
[23] Lin YW, Schuetz AW. In vitro estrogen modulatin of pituitary and
progesterone-induced oocyte maturation in Rana pipients. J Exp Zool, 1983, 226:281-291
[24] Legge M. Oocyte and zygote zona pellucida permeability to macromolecules. J Exp Zool, 1995, 271(2):145-50
[25] Wright CS, Hovatta O, Margara R, et al. Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum Reprod, 1999,14(6): 1555-62
[26] Goud PT, Goud AP, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod, 1998,13(6):1638-1644
[27] 艾继辉，罗丽兰，刘建新．表皮生长因子卵丘细胞对卵母细胞体外成熟的影响．中国实用妇科与产科杂志，2003，19(4)：209-211
[28] 吕述彦，程去英．表皮生长因子调节小鼠生发泡期卵母细胞发育成熟的实验研究．生殖医学杂志，2002，11(1)：25-30
[29] Lorenzo PL, Illera M J, Illera JC, et al. Enhancement of cumulus expansion and nuclear maturation during bovine oocyte maturation in vitro by the addition of epidermal growth factor and insulin-like growth factor I. J Reprod Fertil, 1994, 101(3):697-701
[30] Barnes FL, Sirard MA. Oocyte maturation. Semin Reprod Med, 2000,18(2):123-31
[31] De La Fuente R, O'Brien M J, Eppig JJ. Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes. Hum Reprod., 1999,14(12):3060-8
[32] Mcnatty KP. Follicular fluid. In :Jones, R.E.(Ed.), The Vertebrate Ovary. :lenum Press. New York, 1978:215-259
[33] Ainsworth L, Tsang BK, Downey BR. Interrelationship between follicular fluid steroid levels, gonadotropic stimuli , and oocyte maturation during preovulatory development of porcine follicles. Biol.Reprod, 1980,23(3):621-627
[34] Mingoti GZ, Garcia JM, Rosa-e-Silva AA. The effect of serum on in vitro maturation, fertilization and steroidogenesis of bovine oocytes co-cultured with
granulose cells. Braz J Med Biol Res, 1995, 28(2) :213-217
[35] Moudgal NR, Sherry G, Selvaraj N, et al. Use of a specific aromatase inhibitor for determining whether there is a role for oestrogen in follicle/oocyte maturation, ovulation and preimplantation embryo development. J Reprod Fertil suppl. 1996, 50:69-81
[36] Zheng P, Si W, Bavister B, et al. 17 β-estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys. Hum Reprod, 2003,18(10):2137-2144
[37] Dode MA, Graves, CN. Role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes. Anim Reprod Sci, 2003,78(1-2):99-110
[38] Fukui Y, Fukushima Y, Terawaki Y, et al. Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro. Theriogenology, 1982,18:161-175
[39] Younis AI, Brackett BG, Fayrer-Hosken RA. Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res 1989, 23(2):189-201
[40] McGaughey RW. The culture of pig oocytes in minimal medium, and the influence of progesterone and estradiol-17beta on meiotic maturation. Endocrinology 1977,100(1):39-45
[41] Ochi T. Induction of multiple microtubule-organizing centers, multipolar spindles and multipolar division in cultured V79 cells exposed to diethylstilbestrol, estradiol- 17beta and bisphenol A. Murat Res, 1999, 431 (1): 105-121
[42] Fagbohun CF, Downs SM. Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes. J Reprod Fertil, 1992, 96(2):681-97
[43] Rieger D, Loskutoff NM. Changes in the metabolism of glucose, pyruvate, glutamine and glycine during maturation of cattle oocytes in vitro. J Reprod Fertil, 1994, 100(1):257-62
[44] Downs SM, Utecht AM. Metabolism of radiolabeled glucose by mouse oocytes and
oocyte-cumulus cell complexes. Biol Reprod, 1999,60(6): 1446-52
[45] Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil, 1984, 72(1):9-13
[46] Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci U S A, 1967,58(2):560-7
[47] Donahue RP, Stern S. Follicular cell support of oocyte maturation: production of pyruvate in vitro. J Reprod Fertil, 1968,17(2):395-8
[48] Leese H J, Barton AM. Production of pyruvate by isolated mouse cumulus cells. J Exp Zool,
[49] Downs SM, Mastropolo AM. The participation of energy substrates in the control of meiotic maturation in murine oocytes. Dev Biol, 1994,162(1): 154-68
[50] Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev Biol, 1995, 167(2):502-12
[51] Downs SM, Hudson ER, Hardie DG. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev Biol. 2002,245(1):200-12
[52] Leese HJ. Physiology of the fallopian tube: the provision of nutrients for oocytes and early embryos. In Ovulation to Implantation PP121-125 Eds JHL Evers and MJ Heineman. 1990, Elsevier Science Publishers, New York.
[53] Downs SM, Hudson ED. Energy substrates and the completion of spontaneous meiotic maturation. Zygote, 2000,8(4):339-51
[54] Geshi M, Takenouchi N, Yamauchi N, et al. Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod, 2000, 63(6): 1730-4
[55] Antczak M, Van Blerkom J. Oocyte influences on early evelopment: the regulatory proteins leptin and STAT3 are polarzed in mouse and human oocytes and differentally distributed within the cells of the preimplantation embryo. Mol Hum Reprod, 1997, 3(1):1067-86
[56] 卜淑敏，夏国良，谢辉蓉等．卵丘细胞产生的一氧化氮促进小鼠卵母细胞的成
熟．科学通报，2002，47(22)：1730-1733
[57] Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol, 1988,125(1):181-6
[58] Yoshida M. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol Reprod Dev, 1993,35(1):76-81
[59] Eppig J J, Downs SM. Chemical signals that regulate mammalian oocyte maturation. Biol Reprod, 1996, 54:197-207