【作者】 杨素芳；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2002， 博士

【摘要】 1．系统探讨了放线菌酮（CHX）和细胞松弛素B（CB）对水牛卵母细胞孤雌激活的影响。IVM27～28h的水牛卵母细胞经7％酒精（EH）激活处理5min后，经7％酒精（EH）激活处理5min后，置入不同浓度的（0，0.625，1.25，2.5，5μg／ml）放线菌酮（CHX）培养10h，而后固定染色或在胚胎培养液（CM）中继续培养检查激活分裂率。结果培养在0.625和1.25μg／ml CHX的卵母细胞的激活分裂率和总原核形成率明显高于对照组（P＜0.05），而培养在5μg／mlCHX的卵母细胞的激活分裂率和原核形成率则极显著低于对照组（P＜0.01）。如果在含有0.625μg／ml CHX中添加3μg／ml CB，可明显提高双原核的形成率（38.9％vs15.1％，P＜0.05）和桑椹胚／囊胚发育率（40.7％vs26.8％，P＜0.05），但对其激活分裂率和总原核形成率无明显影响（P＞0.05）。结果表明，适当浓度的CHX能提高卵母细胞激活效果，适当浓度的CB可抑制卵母细胞第二极体的排出，提高孤雌激活后双原核的比例。 2．对水牛卵母细胞的激活方法进行了进一步的研究。IVM26h水牛卵母细胞经7％EH、钙离子载体（A23187）或离子霉素（Ion）激活处理5min后，在2mmol／L6-二甲氨基嘌呤（6-DMAP）中培养3～4h，然后再培养6～11d观察其胚胎发育情况。结果发现，5μmol／L Ion激活处理水牛卵母细胞分裂率（63.0％vs54.2％，P＜0.05）及原核形成率显著高于EH处理的卵母细胞。激活处理前，无Ca²⁺培养液孵育时间的长短，对水牛卵母细胞孤雌发育无明显影响（P＞0.05）。如用A23187激活处理水牛卵母细胞，其激活分裂率随着浓度的升高而提高。结果表明，Ion能非常有效地激活水牛卵母细胞。 3．探讨了受体卵母细胞的成熟和去核时间对水牛核移植效果的影响。卵母细胞IVM16～18h与IVM19～21h的成熟率之间有显著差别（43.0％vs 58.2％，P＜0.05），与22～24h组的成熟率之间有极显著差别（43.0％vs 68.8％，P＜0.01），而采用盲吸法去核的去核率则随着去核时间的延长而显著下降。在体外成熟16～24h期间，水牛卵母细胞去核时间的不同对核移植后的融合率，分裂率和囊胚发育率没有显著影响。结果表明，水牛卵母细胞的成熟率随着成熟时间的延长不断提高，而去核率呈下降趋势，但对其随后的核移植效果没有显著影响（在IVM16～24h期间）。 4．对采用6-DMAP调整去核操作时间的可行性进行了探讨。水牛卵母细胞先经 水牛卵母细胞孤雌激活与体细胞核移植的研究 6－DMAP抑制成熟培养3～sh，再在正常成熟培养液中成熟培养16～18h。 结果发现，与直接在正常成熟培养液中成熟培养16～18h的卵母细胞相比， 其融合率，分裂率和囊胚发育率均无显著影响。山此表明，6oMAP可用于 调整水牛卵母细胞的去核操作时间。5．利用Spindle－View系统对水牛卵母细胞成熟培养时间与细胞核和PBl的位置 关系，以及利用该系统提高去核率的可行性进行了探讨。结果发现，随着卵 母细胞成熟时间的延长，卵母细胞的核与PB；的相对距离增大。体外成熟18h， 20h和 22h的卵母细胞，其细胞核与 PB；位置较近的比例较大（分别为 72石％， 67．9％，64刀％），但当成熟时间增加到 24h时，这一比例下降到 57石％（726％ vs 57石％，P＜0刀5），如采用 Spindle－View系统去核贝可消除这种位置变化对去 核率的影响。由此表明，SPindle－View系统可用于提高卵母细胞的去核率。6．探讨了冷冻保存处理对水牛卵巢颗粒细胞核移植效果的影响。结果发现，颗 粒细胞冷冻保存后再进行核移植的融合率明显下降历7．4％VS 78．7％，P＜0．05人 但随后的分裂率，囊胚发育率无显著变化（P＞0刀5）。由此表明，冷冻保存处 理对细胞核的发育潜能无显著影响，但可能损伤细胞表面的微绒毛，进而影 响细胞间的融合。7．探讨了供体细胞培养处理方法对其核移植效果的影响。水牛颗粒细胞和成体 耳部成纤维细胞经正常培养（DMEM＋10％FCS）、血清饥饿培养 （DMEM＋0．5％FCS 5－10d）或 Aphidicolin（APD）结合血清饥饿培养（0．lqghl APD 24h，0．5％FCS 2叶8d）处理后，分别采用电融合法和直接注核法进行核 移植。结果发现，同一供核细胞各处理组间的核移植胚融合率（以颗粒细胞 作供核）和重组胚的囊胚发育率无显著差异T叩．05入 但经APD门4h)和 0．5％FCS乃～10d)培养处理的供体细胞，其重组胚的分裂率显著高于其它组 （P＜0．05），而其它各组间的分裂率无显著差异T＞队05)。结果表明，血清饥 饿处理水牛供体细胞对其核移植没有显著影响，但DNA合成抑制剂APD结 合血清饥饿培养处理供体细胞，可提高其核移植效果。8．对水牛核移植胚胎的体内发育潜能进行了评定。将来自颗粒细胞和成体成纤 维细胞的12枚核移植胚胎移植到6头受体水牛，其中有一头受孕，5头受体 水牛陆续出现返情。受孕母牛妊娠7个月时，发生流产，产下一s．s ig雄性 死胎(来自22岁的摩拉公牛耳部成纤维细胞人 山此表明，来自高龄公水牛 体细胞的核移植胚胎可以发育到妊娠后期。9．探讨了乙醇预激活水牛成体耳部成纤维细胞对其核移植效果的影更多还原

【Abstract】 1. Effects of cycloheximide (CHX) and cytochalasin B (CB) on the parthenogenetic development of buffalo oocytes were investigated. Buffalo oocytes matured in vitro for 27~28h were treated with 7% EH for 5 min, then cultured in the media containing different concentration of CHX (0, 0.625, 1.25, 2.5, 5ug/ml) for 10h, and then fixed for staining or cultured in CM for checking cleavage. The percentage of oocytes cleaved and formed pronucleus in either 0.625 (Ag/ml or 1.25ug/ml CHX group was significantly higher than the control group (P<0.05). However, when the concentration of CHX was increased to 5 ug/ml, the cleavage rate and pronuclear formation rate were decreased significantly (P<0.01). Addition of 3ug/ml CB to CM containing 0.625 ug/ml CHX increased the proportion of oocytes with two pronuclei (38.9% vs 15.1%, P<0.05) and developed into morulae/blastocysts (40.7% vs 26.8%, P<0.05), but did not affect the percentage of oocytes cleaved and total pronucleus rate. These results indicate that CHX at an appropriate concentration can improve the parthenogenetic development of buffalo oocytes, CB at an appropriate concentration may inhibit the extrusion of the first polar body and increase the proportion of activated oocytes with two pronuclei.2. Methods for activating buffalo oocytes were studied. Buffalo oocytes matured in vitro for 26h were activated by 7% ethanol (EH), A23187 or lonomycin (Ion) for 5min, then cultured in culture medium (CM) containing 2mmol/L for 3-4h, and then cultured in CM for 6-lldays to evaluate embryos development. The cleavage and pronuclear formation rate of oocytes treated with 5umol/L Ion were significantly higher than that of oocytes with EH treatment. Exposing buffalo oocytes to free Ca2+ medium prior to activation treatment did not affect their parthenogenetic development. When buffalo oocytes are activated by A23187, the cleavage increased as the concentrations of A23187 increased. These results indicate that ionomycin can activate buffalo oocytes effectively.3. Effects of in vitro maturation (IVM) duration and enucleation time of buffalo oocytes on the NT embryo development were studied. The maturation rate ofoocytes matured for IVM16-18h was lower than that of IVM for 19-21h (43.0% vs 58.2%, P<0.05), and was significantly lower than that of IVM for 22-24h (43.0% vs 68.8%, P<0.01). However, the enucleation rate decreased as the IVM duration prolonged. There were no significant difference in fusion rate, cleavage rate and blastocyte development rate with different enucleation time of oocytes during IVM16-24h (P>0.05). The results indicate that maturation rate of buffalo oocytes increase as the IVM duration prolongs, while the enucleation rate decreases, however, which has no significant difference on development of NT embryos with different enucleation time of oocytes during IVM16~24h.4. The possibility of adjusting enucleation time by 6-DMAP was explored. Compared with the cytoplasts matured in the maturation medium directly for 16~18h, the fusion rate, cleavage rate and blastocyst development of reconstructed embryos from oocytes cultured in 2mmol/L 6-DMAP for 3-5h prior to IVM did not differ significantly from that of reconstructed embryos from oocytes matured in the maturation medium directly for 16~18h. This result indicates that 6-DMAP may be employed for arranging enucleation of oocytes at a convenient time.5. The position of buffalo oocyte nucleolus related to its PB1 during the final stage of IVM, and the feasibility of increasing enucleation rate using Spindle-View system were studied. The space between PBi and spindle (SP) of IVM buffalo oocytes increased as the IVM duration prolonged. The SP are relatively near to PB1 when oocytes aged at 18h, 20h, 22h (72.6%, 67.9%, 64.0% respectively), when IVM time reached at 24h, the percentage decreased to 57.6%. Enucleation by Spindle-View system could eliminate the effects of changing of the position on enucleation rate. These results indicate that Spindle-View system can increase更多还原