两步体外成熟法对绵羊卵母细胞发育能力的影响
摘要
体外成熟绵羊卵母细胞随后的胚胎发育能力远低于体内成熟的卵母细胞,其主要原因之一是卵母细胞的细胞核成熟早于细胞质成熟,即细胞核与细胞质成熟不同步。本文以绵羊卵母细胞为材料,利用几种核成熟抑制剂组合对其进行两步体外成熟,研究延迟其细胞核成熟进程,目的是提高体外成熟卵母细胞及后期胚胎的发育能力。
首先,采用不同浓度西洛酰胺(Cilostamide)延迟卵母细胞核成熟进程、促卵泡素(FSH)对Cilostamide抑制卵母细胞核成熟,以及Cilostamide抑制卵母细胞核成熟对随后胚胎发育能力的影响进行研究。结果表明:Cilostamide对绵羊卵母细胞核成熟进程的抑制具有剂量依赖性,5μmol/L的Cilostamide为抑制卵母细胞核成熟的适宜浓度,可使绵羊卵母细胞的核成熟进程延长约4 h,而高浓度(10μmol/L和20μmol/L)的Cilostamide将导致部分卵母细胞发生不可逆性抑制;FSH与Cilostamide对绵羊卵母细胞的核成熟起协同抑制作用,成熟液中含有Cilostamide时的卵母细胞体外成熟率随着FSH浓度增加呈现下降趋势;绵羊卵母细胞在含有5μmol/L的Cilostamide的成熟液中体外成熟28 h,然后进行化学激活的囊胚发育率达到(40.4%),显著高于对照组(29.0%) (P <0.05)。
另外,利用3-异丁基-1-甲基黄嘌呤(IBMX)和毛喉素(FSK)两种核成熟抑制剂组合对卵母细胞进行预成熟2 h(第一步成熟),然后转移至含有5μmol/L的Cilostamide成熟液中继续体外成熟28 h(第二步成熟)。其研究结果表明:第一步成熟中的抑制剂组合适宜浓度为100μmol/L IBMX+100μmol/L FSK或100μmol/L IBMX+50μmol/L FSK,囊胚发育率分别达到(51.6%和49.8%),均显著高于对照组(33.1%) (P<0.05);两步体外成熟法所获得囊胚的细胞总数(151.6)与对照组(138)相比,差异不显著(P>0.05)。
第一步成熟处理后COCs中cAMP水平极显著升高,并且高于单独使用FSK与IBMX处理组;第二步成熟过程中,随着成熟时间的延长,实验组及对照组卵母细胞内cAMP水平均呈降低趋势,在成熟第9 h、20 h时对照组卵母细胞中cAMP水平已降至较低而实验组卵母细胞中cAMP仍然保持较高水平。到30 h时实验组与对照组卵母细胞内cAMP水平相比,差异不显著(P>0.05)。
At present, the subsequent embryo development ability of ovine oocytes maturated in vitro is far lower than the embryos derived from in vivo matured oocytes, the main reason may be the nuclear maturation earlier than cytoplasmic maturation. It is to say oocyte nuclear and cytoplasmic maturation is not synchronous. In this paper, ovine oocytes were matured by two step mature method which using a variety of nuclear maturation inhibitors, delayed oocyte nuclear maturation, in order to make the oocyte nuclear and cytoplasmic matured as synchronous as possible, so as to improve the quality of oocytes matured in vitro and subsequent embryo development competence.
At first, researched the different concentrations of Cilostamide delayed oocyte nuclear maturation time and inhibit effect, the influence of Follicle stimulating hormone (FSH) on Cilostamide maturation inhibition and the effect of Cilostamide inhibits oocyte nuclear maturation on subsequent embryonic development ability. The study shows that Cilostamide on sheep oocyte nuclear maturation inhibition in a dose-dependent manner, and the 5μmol/L Cilostamide is the suitable concentration for sheep oocyte maturation in vitro, the oocyte nuclear maturation can be delayed about 4 hours. While the high concentration (10μmol/L and 20μmol/L) Cilostamide will lead to some oocytes lost the ability to resume meiosis. FSH shows synergistic effect on Cilostamide inhibited oocyte nuclear maturation. When Cilostamide exists, with FSH concentration increase, the oocyte maturation rate of each group showed a downward trend. Ovine oocytes treaded with 5μmol/L Cilostamide 28 h after chemical activation, the blastocyst rate was 40.4% higher than that in the control group 29.0% (P<0.05).
In addition, oocyte were treaded during per-IVM 2 h (the first maturation step) with the culture medium contained IBMX and FSK for, then transfered to the culture medium contained 5μmol/L Cilostamide matured in vitro (the second maturation step). The study shows that 100μmol/L IBMX+100μmol/L FSK or 100μmol/L+100μmol/L FSK was the better inhibitor combination choosed for the first step, the blastocyst rate were (51.6% and 49.8%) higher than that in the control group (33.1%) (P<0.05). Compared with the control group,the blastocyst cell number of two step group (151.6vs138) has no significant difference (P>0.05), so that two step maturation in vitro does not reduce the blastocyst cell number.
After the first step mature treatment, cAMP levels were significantly increased in COCs and higher than that of FSK alone and IBMX treatment group. With the extension of maturity time, both the control group and experimental group, oocyte cAMP levels are decreased. At the IVM 9 h、20 h, the cAMP levels of control group oocyte have been reduced to lower,but the cAMP level of experimental group oocytes remained high level. At IVM 30 h,the cAMP level of two step group and the control group was no longer significant.
首先,采用不同浓度西洛酰胺(Cilostamide)延迟卵母细胞核成熟进程、促卵泡素(FSH)对Cilostamide抑制卵母细胞核成熟,以及Cilostamide抑制卵母细胞核成熟对随后胚胎发育能力的影响进行研究。结果表明:Cilostamide对绵羊卵母细胞核成熟进程的抑制具有剂量依赖性,5μmol/L的Cilostamide为抑制卵母细胞核成熟的适宜浓度,可使绵羊卵母细胞的核成熟进程延长约4 h,而高浓度(10μmol/L和20μmol/L)的Cilostamide将导致部分卵母细胞发生不可逆性抑制;FSH与Cilostamide对绵羊卵母细胞的核成熟起协同抑制作用,成熟液中含有Cilostamide时的卵母细胞体外成熟率随着FSH浓度增加呈现下降趋势;绵羊卵母细胞在含有5μmol/L的Cilostamide的成熟液中体外成熟28 h,然后进行化学激活的囊胚发育率达到(40.4%),显著高于对照组(29.0%) (P <0.05)。
另外,利用3-异丁基-1-甲基黄嘌呤(IBMX)和毛喉素(FSK)两种核成熟抑制剂组合对卵母细胞进行预成熟2 h(第一步成熟),然后转移至含有5μmol/L的Cilostamide成熟液中继续体外成熟28 h(第二步成熟)。其研究结果表明:第一步成熟中的抑制剂组合适宜浓度为100μmol/L IBMX+100μmol/L FSK或100μmol/L IBMX+50μmol/L FSK,囊胚发育率分别达到(51.6%和49.8%),均显著高于对照组(33.1%) (P<0.05);两步体外成熟法所获得囊胚的细胞总数(151.6)与对照组(138)相比,差异不显著(P>0.05)。
第一步成熟处理后COCs中cAMP水平极显著升高,并且高于单独使用FSK与IBMX处理组;第二步成熟过程中,随着成熟时间的延长,实验组及对照组卵母细胞内cAMP水平均呈降低趋势,在成熟第9 h、20 h时对照组卵母细胞中cAMP水平已降至较低而实验组卵母细胞中cAMP仍然保持较高水平。到30 h时实验组与对照组卵母细胞内cAMP水平相比,差异不显著(P>0.05)。
At present, the subsequent embryo development ability of ovine oocytes maturated in vitro is far lower than the embryos derived from in vivo matured oocytes, the main reason may be the nuclear maturation earlier than cytoplasmic maturation. It is to say oocyte nuclear and cytoplasmic maturation is not synchronous. In this paper, ovine oocytes were matured by two step mature method which using a variety of nuclear maturation inhibitors, delayed oocyte nuclear maturation, in order to make the oocyte nuclear and cytoplasmic matured as synchronous as possible, so as to improve the quality of oocytes matured in vitro and subsequent embryo development competence.
At first, researched the different concentrations of Cilostamide delayed oocyte nuclear maturation time and inhibit effect, the influence of Follicle stimulating hormone (FSH) on Cilostamide maturation inhibition and the effect of Cilostamide inhibits oocyte nuclear maturation on subsequent embryonic development ability. The study shows that Cilostamide on sheep oocyte nuclear maturation inhibition in a dose-dependent manner, and the 5μmol/L Cilostamide is the suitable concentration for sheep oocyte maturation in vitro, the oocyte nuclear maturation can be delayed about 4 hours. While the high concentration (10μmol/L and 20μmol/L) Cilostamide will lead to some oocytes lost the ability to resume meiosis. FSH shows synergistic effect on Cilostamide inhibited oocyte nuclear maturation. When Cilostamide exists, with FSH concentration increase, the oocyte maturation rate of each group showed a downward trend. Ovine oocytes treaded with 5μmol/L Cilostamide 28 h after chemical activation, the blastocyst rate was 40.4% higher than that in the control group 29.0% (P<0.05).
In addition, oocyte were treaded during per-IVM 2 h (the first maturation step) with the culture medium contained IBMX and FSK for, then transfered to the culture medium contained 5μmol/L Cilostamide matured in vitro (the second maturation step). The study shows that 100μmol/L IBMX+100μmol/L FSK or 100μmol/L+100μmol/L FSK was the better inhibitor combination choosed for the first step, the blastocyst rate were (51.6% and 49.8%) higher than that in the control group (33.1%) (P<0.05). Compared with the control group,the blastocyst cell number of two step group (151.6vs138) has no significant difference (P>0.05), so that two step maturation in vitro does not reduce the blastocyst cell number.
After the first step mature treatment, cAMP levels were significantly increased in COCs and higher than that of FSK alone and IBMX treatment group. With the extension of maturity time, both the control group and experimental group, oocyte cAMP levels are decreased. At the IVM 9 h、20 h, the cAMP levels of control group oocyte have been reduced to lower,but the cAMP level of experimental group oocytes remained high level. At IVM 30 h,the cAMP level of two step group and the control group was no longer significant.
引文
[1]Watson AJ. Oocyte cytoplasmic maturation: A key mediator of oocyte and embryo development competence[J]. Anim Sci, 2007, 85(13): E1～3.
[2]Lonergan P, Monaghan P, Rizos D, et al. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro[J]. Mol Reprod Dev, 1994, 37(1):48～53.
[3]Sirad M A, Coenen K and Bilodeau S. Effects of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes[J]. Theriogenology, 1992, 37(6):39～57.
[4]Calderon I, and Healy D. Handbook of In vitro Fertilization[M]. BocaRaton, Florida: CRC Press, 1993, 1～16.
[5]Alm H, Tormer H, Lohrke B et al. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresylblue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity[J]. Theriogenology, 2005, 63(8):2194～2205.
[6]Greve T, Xu KP, Callesen H, et al. In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro[J]. J In Vitro Fertil Embryo Transf, 1987, 4(5):281～285.
[7]Leibfried-Rutledge ML, Critser ES, Eyestone WH, et al. Development potential of bovine oocytes matured in vitro or in vivo[J]. Biol Reprod 1987, 36(3):376～383.
[8]Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence[J]. Reproduction 2001; 121(1):51～75.
[9]葛宝生,梁冠生,刘建民等.绵羊卵泡体外成熟的研究[J].兽医大学学报, 1993, 13 (1): 66～691.
[10]旭日干,张锁链,薛晓先等.屠宰母牛卵巢卵母细胞的体外受精与早期发生[J].内蒙古大学学报,1989, 20 (3):407～414.
[11]旭日干.体外成熟、体外受精的绵羊胚胎的发育和手术移植的成绩[J].家畜繁殖学, 1990, 36(4):221.
[12]Aktas H, Wheeler MB, Rosenkrans CF Jr, et al. Maintenance of bovine oocytes in prophase of MI by high cAMP[J]. J Reprod Fertil, 1995, 105 (2):227～235.
[13]Braun J. Influence of protein supplement and culture conditions on cumulus cell expansion and nuclear maturation of sheep follicular oocytes[J]. Theriogenology, 1988 , 29 (2):200～202.
[14]李永海,刘瑞华,焦丽红等.哺乳动物卵母细胞核质成熟的差异及其影响因素[J].农业生物技术学报, 2003, 11(1):94～100.
[15]Van Blerkom J. Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes[J]. Proc Natl Acad Sci USA, 1991, 88(11): 5031～5035.
[16]李荣凤,薛晓先,刘哲等.牛卵母细胞皮层颗粒荧光染色法质成熟鉴定[J].畜牧兽医学报, 2000, 31(3) : 203～210.
[17]Niw a K, Park C-K, Okuda K. Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa[J]. Reprod Fert, 1991, 91(1): 329～336.
[18]Velilla E, Izquierdo D, Rodriguez Gonzalez E, et al. Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilization: ultrastructural and cytoche -mical study[J]. Mol Reprod Dev, 2004, 68(4): 507～514.
[19]Thompson JG, McGowan LT, Tervit HR. Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos culture in vitro[J]. J Reprod Fertil, 2000, 118(1): 47～55.
[20]Stojkovic M, Machado SA, Stojkovic P, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture[J]. Biol Reprod, 2001, 64(3):904～909.
[21]Nishi Y, Takeshita T, Sato K, et al. Change of the mitochondrial distribution in mouse ooplasm during in vitro maturation[J]. J Nippon Med Sch, 2003, 70(5): 408～415.
[22]陈娟,冯婷,丁卫等.人卵母细胞的形态对受精及早期胚胎发育的影响[J].医学研究生学报, 2004, 17(4): 289～291.
[23]Hansen D V, Pomerening JR, Summers MK, et al. Emi2 at the crossroads: where CSF meets MPF[J]. Cell Cycle, 2007, 6(6): 732-738.
[24]Ohe M, Inoue D, Kanemori Y, et al. Erp1 /Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes[J]. Dev Biol, 2007, 303(1): 157～160.
[25]Ito J, Kato M, Hochi S, et al. Effect of enucleation on inactivation of cytostatic factor activity in matured rat oocytes[J]. Cloning Stem Cells, 2007, 9(2): 257～266.
[26]Downs S M. Purine control of mouse oocytes maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest[J]. Mol Reprod Dev, 1993, 35(1): 82～94.
[27]Kanatsu-Shinohara M, Schultz RM and Kopf GS. Acquisition of meiotic competence in mouse oocytes:absolute amounts of p34,cdc2,cyclin B1,cdc25C,and wee1 in meiotically incompetent and competent oocytes[J]. Biology of Reproduction, 2000, 63(6):1610～1616.
[28]Tremoleda JI, Tharanit T, Van Tol HT, et a1. Effects of follicu1ar cells and FSH on the resumption of meios is in equine oocytes matured in vitro[J]. Reproduction, 2003, 125 (4):565～570.
[29]Kim JS, Cho YS, Song BS, et al. Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation;itstimulates further embryonic development including blastocyst quality in pigs[J]. Theriogenology, 2008, 69(3):290～301.
[30]Shu YM, Zeng HT, Ren Z, et al. Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocyte[J]. Hum Reprod,2008,23(3):504～513
[31]Conti M, Andersen CB, Richard FJ, et al. Role of cyclic nucleotide phosphodiesterases in resumption of meiosis[J]. Mol Cell Endocrinol, 1998, 145(2): 9～14
[32]Honrer, K. Rodent oocytes exPress an active daenylyl cyclase erquired for meiotic arrest[J]. Dev Biol, 2003, 258(2):385～396.
[33]曹文,潘玲梅,田谷一善.磷酸二酯酶在卵母细胞成熟中的作用[J].细胞生物学杂志, 2009, 31(4): 497～502.
[34]Mehlmann L M, Saeki Y, Tanaka S. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes[J]. Science, 2004, 306(5703):1947～1950.
[35]Bedner P, Niessen H, Odermatt B, et al. Selective permeability of different connexin channels to the second messenger cyclic AMP[J]. J Biol Chem, 2006, 281(10): 6673～6681.
[36]Omori K, K otera J. Overview of PDEs and their regulation[J]. Circ Res.2007, 100 (3):309～327.
[37]陈勇,夏国良,苏友强等.体外培养条件下促性腺激素对昆明白小鼠卵母细胞成熟及卵丘扩展的影响[J].动物学报, 2001, 47 (2): 203～208.
[38]傅国栋,张似青,夏国良. Forskolin和促性腺激素对猪卵母细胞体外成熟的影响[J].上海农业学报, 2002, 18(1): 84～87.
[39]Xia, G L. Effect of mouse age and gap junction breaking of cumulus oocyte complexes on oocyt e maturation in vitro[J]. Acta Zool Sin, 42(4): 408～413.
[40]曲杨燕,赵雁伟,李凯等. FSH对绵羊卵母细胞体外核成熟的影响[J].中国畜牧兽医, 2008, 35 (12):80～83.
[41]Mattioli M, Barboni B. Calcium eievation in sheep cumulus-oocyte complexes after luteinizing hormone stimulation[J]. Mol Repro Dev, 1998, 50(3):361～369.
[42]Anderson CY, Baltsen M, Byskov AG. Gonadotropin-induced resumption of oocyte meiosis and meiosis-activating sterols [J]. Curr Top Dev Biol, 1999, 41:163～185.
[43]桑润滋.动物繁殖生物技术[M].北京:中国农业出版社, 2002, 6.
[44]张晓华,王克钦.体外培养牛卵母细胞的核成熟与质成熟[J].黑龙江动物繁殖, 2007, 15(3): 15～17.
[45]Sirard MA, Florman HM, Lebfried-Rutledge ML. Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes[J]. Biol Reprod, 1989, 40(6): 1257～1263.
[46]钱云,刘红林.卵母细胞成熟的形态学特征及其调控[J].国外畜牧科技, 1999, 26 (3):26～28.
[47]陈慧,苏迎春,孙迎璞.线粒体在卵子和早期胚胎发育中的研究[J].国外医学计划生育生殖健康分册, 2007, 26(2):60～63.
[48]Cao X, Zhou P, Luo HL, et al. The effect of VEGF on the temporal-spatial change of alpha-tubulin and cortical granules of ovine oocytes matured in vitro[J]. Anim Reprod Sci, 2009, 113(1-4):236～250.
[49]Hosoe M, Shioya Y. Distribution of cortical granules in classified bovine oocyte[J]. Zygote, 1997, 5(4):371～376.
[50]Poulton J, Marchington DR. Segregation of mitochondrial DNA(mt DNA) in human oocytes and in animal models of mtDNA disease: clinical implications[J]. Reproduction, 2002, 123(6): 751～755.
[51]张玲.卵母细胞成熟抑制与发育潜力[J].生殖医学, 2004, 13(4): 252～254.
[52]Conti M, Richter W, Mehats C, et al. Cyclie AMP-specific PDE4 PhosPhodiesterase as critical comPonents of eyelie AMP signaling[J]. Biol Chezn, 2003, 278(8):5493～5496.
[53]Stefall E, Wiesner B, Balllie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells[J]. J Am Soe NePhrol, 2007, 18(l): 199～212.
[54]Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A in rat oocyte maturation[J]. Biol Reprod, 2000, 65(3):1444～1451.
[55]Maxime Sasseville, et al. 3’5’-Cyclic Adenosine Monophosphate-Dependent Up-Regulation of Phosphodiesterase Type 3A in Porcine CumulusCells[J]. Endorinol, 2007, 148(4): 1858～1867.
[56]Thomas RE, Thompson JG, Armstrong DT, et al. Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity[J]. Biol Reprod, 2004, 71(4): 1142～1149.
[57]周波,夏国良,旭日干等.绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响[D].北京:中国农业大学, 2005.
[58]Leen Vanhoutte, Petra De Sutter, Daniela Nogueira, et al. Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase3 inhibitor[J]. Hum Reprod, 2007, 22(5):1239～1246.
[59]Nogueira D, Cortvrindt R, De Matos DG, et al. Effect of phosphodiesterase type 3 inbibitor on developmental competence of immature mouse oocytes in vitro[J]. Biol Reprod, 2003, 69(6):2045～2052.
[60]周红林,马峻,季维智.绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响[J].动物学报, 2002, 48 (6) :741～748
[61] Fouladi N A A. Maintenance of bovine oocytes in meiotic arrest and subsequent development in vitro: acomparative evaluation of antral-follicle culture with other methods[J]. Biol Reprod, 1998, 59(2):255～262.
[62]雷安居,高志敏,杨春荣等.牛卵母细胞的体外成熟培养[J].西北农林科技大学学报(自然科学版) , 2002, 30 (4):95～97.
[63]卜淑敏,周波,夏国良等. 3种磷酸二酯酶对小鼠卵母细胞自发成熟的影响[J].中国农业大学学报, 2007, 12 (5): 1～4.
[64]Yi-min Shu, Hai-tao Zeng, Zi Ren, et a1. Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes[J]. Hum Reprod, 2008, 23(3):504～513.
[65]Kim J, Cho Y, Song B, et al. Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation;it stimulates further embryonic development including blastocyst quality in pigs[J]. Theriogenology, 2008, 69(3):290～301.
[66]陈大元.受精生物学-受精机制与生殖工程[M].北京:科学出版社, 2000.
[67]孙桂金,尹逊河,敖红等.影响绵羊体外受精技术因素的研究[J].中国畜牧与兽医, 2006, 2(33):30～32.
[68]Nogueira D, Cortvrindt R, De Matos DG, et al. Effect ofphosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro[J]. Biol Reprod, 2003, 69(6): 2045～2052.
[69]Tanghe S, Vansoom A, Nauwynck H, et al. Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization[J]. Mol Reprod dev, 2002, 61(3): 414～424.
[70]Rebecca E, Thomas, David T, et al. Differential Effects of Specific Phosphodiesterase -Isoenzyme Inhibitors on Bovine Oocyte Meiotic Maturation[J]. Dev Biol, 2002, 244 (2):215～225.
[2]Lonergan P, Monaghan P, Rizos D, et al. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro[J]. Mol Reprod Dev, 1994, 37(1):48～53.
[3]Sirad M A, Coenen K and Bilodeau S. Effects of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes[J]. Theriogenology, 1992, 37(6):39～57.
[4]Calderon I, and Healy D. Handbook of In vitro Fertilization[M]. BocaRaton, Florida: CRC Press, 1993, 1～16.
[5]Alm H, Tormer H, Lohrke B et al. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brilliant cresylblue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity[J]. Theriogenology, 2005, 63(8):2194～2205.
[6]Greve T, Xu KP, Callesen H, et al. In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro[J]. J In Vitro Fertil Embryo Transf, 1987, 4(5):281～285.
[7]Leibfried-Rutledge ML, Critser ES, Eyestone WH, et al. Development potential of bovine oocytes matured in vitro or in vivo[J]. Biol Reprod 1987, 36(3):376～383.
[8]Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence[J]. Reproduction 2001; 121(1):51～75.
[9]葛宝生,梁冠生,刘建民等.绵羊卵泡体外成熟的研究[J].兽医大学学报, 1993, 13 (1): 66～691.
[10]旭日干,张锁链,薛晓先等.屠宰母牛卵巢卵母细胞的体外受精与早期发生[J].内蒙古大学学报,1989, 20 (3):407～414.
[11]旭日干.体外成熟、体外受精的绵羊胚胎的发育和手术移植的成绩[J].家畜繁殖学, 1990, 36(4):221.
[12]Aktas H, Wheeler MB, Rosenkrans CF Jr, et al. Maintenance of bovine oocytes in prophase of MI by high cAMP[J]. J Reprod Fertil, 1995, 105 (2):227～235.
[13]Braun J. Influence of protein supplement and culture conditions on cumulus cell expansion and nuclear maturation of sheep follicular oocytes[J]. Theriogenology, 1988 , 29 (2):200～202.
[14]李永海,刘瑞华,焦丽红等.哺乳动物卵母细胞核质成熟的差异及其影响因素[J].农业生物技术学报, 2003, 11(1):94～100.
[15]Van Blerkom J. Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes[J]. Proc Natl Acad Sci USA, 1991, 88(11): 5031～5035.
[16]李荣凤,薛晓先,刘哲等.牛卵母细胞皮层颗粒荧光染色法质成熟鉴定[J].畜牧兽医学报, 2000, 31(3) : 203～210.
[17]Niw a K, Park C-K, Okuda K. Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa[J]. Reprod Fert, 1991, 91(1): 329～336.
[18]Velilla E, Izquierdo D, Rodriguez Gonzalez E, et al. Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilization: ultrastructural and cytoche -mical study[J]. Mol Reprod Dev, 2004, 68(4): 507～514.
[19]Thompson JG, McGowan LT, Tervit HR. Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos culture in vitro[J]. J Reprod Fertil, 2000, 118(1): 47～55.
[20]Stojkovic M, Machado SA, Stojkovic P, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture[J]. Biol Reprod, 2001, 64(3):904～909.
[21]Nishi Y, Takeshita T, Sato K, et al. Change of the mitochondrial distribution in mouse ooplasm during in vitro maturation[J]. J Nippon Med Sch, 2003, 70(5): 408～415.
[22]陈娟,冯婷,丁卫等.人卵母细胞的形态对受精及早期胚胎发育的影响[J].医学研究生学报, 2004, 17(4): 289～291.
[23]Hansen D V, Pomerening JR, Summers MK, et al. Emi2 at the crossroads: where CSF meets MPF[J]. Cell Cycle, 2007, 6(6): 732-738.
[24]Ohe M, Inoue D, Kanemori Y, et al. Erp1 /Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes[J]. Dev Biol, 2007, 303(1): 157～160.
[25]Ito J, Kato M, Hochi S, et al. Effect of enucleation on inactivation of cytostatic factor activity in matured rat oocytes[J]. Cloning Stem Cells, 2007, 9(2): 257～266.
[26]Downs S M. Purine control of mouse oocytes maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest[J]. Mol Reprod Dev, 1993, 35(1): 82～94.
[27]Kanatsu-Shinohara M, Schultz RM and Kopf GS. Acquisition of meiotic competence in mouse oocytes:absolute amounts of p34,cdc2,cyclin B1,cdc25C,and wee1 in meiotically incompetent and competent oocytes[J]. Biology of Reproduction, 2000, 63(6):1610～1616.
[28]Tremoleda JI, Tharanit T, Van Tol HT, et a1. Effects of follicu1ar cells and FSH on the resumption of meios is in equine oocytes matured in vitro[J]. Reproduction, 2003, 125 (4):565～570.
[29]Kim JS, Cho YS, Song BS, et al. Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation;itstimulates further embryonic development including blastocyst quality in pigs[J]. Theriogenology, 2008, 69(3):290～301.
[30]Shu YM, Zeng HT, Ren Z, et al. Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocyte[J]. Hum Reprod,2008,23(3):504～513
[31]Conti M, Andersen CB, Richard FJ, et al. Role of cyclic nucleotide phosphodiesterases in resumption of meiosis[J]. Mol Cell Endocrinol, 1998, 145(2): 9～14
[32]Honrer, K. Rodent oocytes exPress an active daenylyl cyclase erquired for meiotic arrest[J]. Dev Biol, 2003, 258(2):385～396.
[33]曹文,潘玲梅,田谷一善.磷酸二酯酶在卵母细胞成熟中的作用[J].细胞生物学杂志, 2009, 31(4): 497～502.
[34]Mehlmann L M, Saeki Y, Tanaka S. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes[J]. Science, 2004, 306(5703):1947～1950.
[35]Bedner P, Niessen H, Odermatt B, et al. Selective permeability of different connexin channels to the second messenger cyclic AMP[J]. J Biol Chem, 2006, 281(10): 6673～6681.
[36]Omori K, K otera J. Overview of PDEs and their regulation[J]. Circ Res.2007, 100 (3):309～327.
[37]陈勇,夏国良,苏友强等.体外培养条件下促性腺激素对昆明白小鼠卵母细胞成熟及卵丘扩展的影响[J].动物学报, 2001, 47 (2): 203～208.
[38]傅国栋,张似青,夏国良. Forskolin和促性腺激素对猪卵母细胞体外成熟的影响[J].上海农业学报, 2002, 18(1): 84～87.
[39]Xia, G L. Effect of mouse age and gap junction breaking of cumulus oocyte complexes on oocyt e maturation in vitro[J]. Acta Zool Sin, 42(4): 408～413.
[40]曲杨燕,赵雁伟,李凯等. FSH对绵羊卵母细胞体外核成熟的影响[J].中国畜牧兽医, 2008, 35 (12):80～83.
[41]Mattioli M, Barboni B. Calcium eievation in sheep cumulus-oocyte complexes after luteinizing hormone stimulation[J]. Mol Repro Dev, 1998, 50(3):361～369.
[42]Anderson CY, Baltsen M, Byskov AG. Gonadotropin-induced resumption of oocyte meiosis and meiosis-activating sterols [J]. Curr Top Dev Biol, 1999, 41:163～185.
[43]桑润滋.动物繁殖生物技术[M].北京:中国农业出版社, 2002, 6.
[44]张晓华,王克钦.体外培养牛卵母细胞的核成熟与质成熟[J].黑龙江动物繁殖, 2007, 15(3): 15～17.
[45]Sirard MA, Florman HM, Lebfried-Rutledge ML. Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes[J]. Biol Reprod, 1989, 40(6): 1257～1263.
[46]钱云,刘红林.卵母细胞成熟的形态学特征及其调控[J].国外畜牧科技, 1999, 26 (3):26～28.
[47]陈慧,苏迎春,孙迎璞.线粒体在卵子和早期胚胎发育中的研究[J].国外医学计划生育生殖健康分册, 2007, 26(2):60～63.
[48]Cao X, Zhou P, Luo HL, et al. The effect of VEGF on the temporal-spatial change of alpha-tubulin and cortical granules of ovine oocytes matured in vitro[J]. Anim Reprod Sci, 2009, 113(1-4):236～250.
[49]Hosoe M, Shioya Y. Distribution of cortical granules in classified bovine oocyte[J]. Zygote, 1997, 5(4):371～376.
[50]Poulton J, Marchington DR. Segregation of mitochondrial DNA(mt DNA) in human oocytes and in animal models of mtDNA disease: clinical implications[J]. Reproduction, 2002, 123(6): 751～755.
[51]张玲.卵母细胞成熟抑制与发育潜力[J].生殖医学, 2004, 13(4): 252～254.
[52]Conti M, Richter W, Mehats C, et al. Cyclie AMP-specific PDE4 PhosPhodiesterase as critical comPonents of eyelie AMP signaling[J]. Biol Chezn, 2003, 278(8):5493～5496.
[53]Stefall E, Wiesner B, Balllie GS, et al. Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells[J]. J Am Soe NePhrol, 2007, 18(l): 199～212.
[54]Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A in rat oocyte maturation[J]. Biol Reprod, 2000, 65(3):1444～1451.
[55]Maxime Sasseville, et al. 3’5’-Cyclic Adenosine Monophosphate-Dependent Up-Regulation of Phosphodiesterase Type 3A in Porcine CumulusCells[J]. Endorinol, 2007, 148(4): 1858～1867.
[56]Thomas RE, Thompson JG, Armstrong DT, et al. Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity[J]. Biol Reprod, 2004, 71(4): 1142～1149.
[57]周波,夏国良,旭日干等.绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响[D].北京:中国农业大学, 2005.
[58]Leen Vanhoutte, Petra De Sutter, Daniela Nogueira, et al. Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase3 inhibitor[J]. Hum Reprod, 2007, 22(5):1239～1246.
[59]Nogueira D, Cortvrindt R, De Matos DG, et al. Effect of phosphodiesterase type 3 inbibitor on developmental competence of immature mouse oocytes in vitro[J]. Biol Reprod, 2003, 69(6):2045～2052.
[60]周红林,马峻,季维智.绵羊卵母细胞体外核成熟抑制及其对胚胎发育潜力的影响[J].动物学报, 2002, 48 (6) :741～748
[61] Fouladi N A A. Maintenance of bovine oocytes in meiotic arrest and subsequent development in vitro: acomparative evaluation of antral-follicle culture with other methods[J]. Biol Reprod, 1998, 59(2):255～262.
[62]雷安居,高志敏,杨春荣等.牛卵母细胞的体外成熟培养[J].西北农林科技大学学报(自然科学版) , 2002, 30 (4):95～97.
[63]卜淑敏,周波,夏国良等. 3种磷酸二酯酶对小鼠卵母细胞自发成熟的影响[J].中国农业大学学报, 2007, 12 (5): 1～4.
[64]Yi-min Shu, Hai-tao Zeng, Zi Ren, et a1. Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes[J]. Hum Reprod, 2008, 23(3):504～513.
[65]Kim J, Cho Y, Song B, et al. Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation;it stimulates further embryonic development including blastocyst quality in pigs[J]. Theriogenology, 2008, 69(3):290～301.
[66]陈大元.受精生物学-受精机制与生殖工程[M].北京:科学出版社, 2000.
[67]孙桂金,尹逊河,敖红等.影响绵羊体外受精技术因素的研究[J].中国畜牧与兽医, 2006, 2(33):30～32.
[68]Nogueira D, Cortvrindt R, De Matos DG, et al. Effect ofphosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro[J]. Biol Reprod, 2003, 69(6): 2045～2052.
[69]Tanghe S, Vansoom A, Nauwynck H, et al. Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization[J]. Mol Reprod dev, 2002, 61(3): 414～424.
[70]Rebecca E, Thomas, David T, et al. Differential Effects of Specific Phosphodiesterase -Isoenzyme Inhibitors on Bovine Oocyte Meiotic Maturation[J]. Dev Biol, 2002, 244 (2):215～225.