【作者】 胡媛媛；

【导师】 田树军；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2012， 硕士

【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)。更多还原

【Abstract】 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.更多还原