摘要
盘羊是一种珍稀的野生保护动物,长期的过度猎杀是盘羊面临灭绝的主要因素,利用异种核移植技术来保护濒危动物极具发展潜力。本研究通过体外培养技术建立了盘羊的耳成纤维细胞系,以此为供体,绵羊卵母细胞为受体,利用显微操作技术构建了异种重构胚,通过对核移植各技术环节的优化,显著提高了胚胎的体外发育率,且将重构胚移植到代孕绵羊体内,其中有2只受体羊延迟到90 d后返情,最终未获得妊娠。
1供体细胞系的建立
采取新疆野生盘羊耳皮肤组织和新疆本地绵羊流产胎儿的皮肤组织,利用组织块培养法建立了盘羊和绵羊成纤维细胞系;由体外成熟的绵羊卵巢卵丘-卵母细胞复合体获取卵丘细胞,进行原代传代培养.通过对盘羊成纤维细胞、绵羊成纤维细胞和卵丘细胞三种细胞系的细胞生长曲线测定,发现盘羊皮肤成纤维细胞的潜伏期比绵羊成纤维细胞和卵丘细胞的潜伏期长,没有明显的平台期。生长曲线波动规则,符合成纤维细胞的体外生长特征,适合作为核移植供体细胞。
为建立适合盘羊成纤维细胞的冷冻保存体系,本文研究了冷冻程序和冷冻液对盘羊皮肤成纤维细胞冷冻存活率的影响。选用3种不同的冷冻方法,程序1(先4℃冰箱预冷30 min,-20℃放置3 h,-70℃过夜,然后投入液氮中保存)冷冻细胞解冻后的存活率为95.2%,细胞的贴壁率在接种18 h后达到70%～80%,显著高于其它两个冷冻程序。在程序1中,将细胞在-20℃保存3～5 h,没有显著影响细胞的存活率,接种后细胞的生长状况也没有明显的差异。采用冷冻液1(50%FBS)对细胞进行冷冻,解冻后细胞的存活率显著高于冷冻液2(20%FBS)组,且前者细胞解冻接种的贴壁率也稍高于后者。结果表明,利用程序1,采用50%FBS的冷冻液对细胞进行冷冻保存,可以得到较高的解冻后细胞存活率。
运用常规的染色体核型分析技术,对新疆珍稀野生盘羊和当地绵羊的染色体核型进行研究。结果表明:盘羊的染色体数2n=56,其中有27对常染色体和1对性染色体,1号和2号常染色体为中着丝粒染色体,其余的3～27号为端着丝粒染色体;X染色体为最大的端着丝粒染色体,Y染色体为最小的中着丝粒染色体.绵羊的二倍体染色体数2n=54,其中包括26对常染色体和1对性染色体,常染色体中1、2和3号为中着丝粒染色体,4～26号为端着丝粒染色体;X染色体为最大的端着丝粒染色体,Y染色体为最小的中着丝粒染色体。
2盘羊异种重构胚的构建
本试验比较了绵羊卵巢不同的保存温度、时间与保存液对卵母细胞孤雌发育能力的影响。结果表明,14～18℃保存15～17h的卵巢卵母细胞的成熟率(80.9%vs 82.2%)、孤雌激活后囊胚的发育率(21.0%vs 22.0%)与对照组间均无显著差异,采用生理盐水或D-PBS保存卵巢对卵母细胞的发育亦无显著影响;而将卵巢在4℃或25～30℃保存15～17 h却显著降低了卵母细胞的成熟率(61.9%vs 19%)与卵裂率(8.6%vs 9.1%),没有发育到囊胚。从而得出,14～18℃的保存温度对卵巢的长时间保存是比较适宜的。
受体卵母细胞去核是核移植关键步骤之一。盲吸法是最常采用的去核方法,化学辅助去核法则是利用秋水仙素处理卵母细胞,从而使卵母细胞形成一个包含浓缩染色体的胞质突起,利用去核针将其吸除,以达到去核目的。本文比较了在化学辅助去核法中,秋水仙素的浓度争作用时间对显示效果的影响;且对两种去核法的去核效率进行了探讨。结果表明:(1)成熟的卵母细胞在0.4μg/mL的秋水仙素溶液中分别孵育0.5 h和1 h,胞质突起率和去核率没有显著的差异,突起率可高达85.4%,去核率达到100%.(2)分别用0.2μg/mL和0.4μg/mL秋水仙素对卵母细胞处理0.5h,均能达到有效去核的目的。(3)卵母细胞体外成熟18～23 h,不会影响秋水仙素诱导胞质突起的比率和去核率;且对成熟21～23 h的卵母细胞采用化学辅助去核法去核,去核率显著高于盲吸去核法.(4)对成熟18～20 h的卵母细胞进行盲吸法或化学辅助法去核,重构胚的发育率没有显著差异。由此可见,化学辅助去核法(0.2μg/mL的秋水仙素处理0.5 h)可以高效快速地去核,且不影响重构胚的早期发育。
另外,本试验还比较了不同的电融合程序、激活方法和胚胎培养液对异种重构胚发育的影响。结果显示:异种重构胚的适宜电融合参数为2个121～125 V/mm、40μs/次的直流电脉冲或2个126～130 V/mm、20μs/次的直流电脉冲;融合后,对未融合的重构胚以同样的融合参数再融合一次,不会影响重构胚的发育率,可得到更多的可用异种核移植胚胎。卵母细胞成熟培养25 h和26 h激活组的囊胚率显著高于成熟培养24 h激活组:6-DMAP激活液作用2 h,激活效果较好;分别采用EH+6-DMAP或Iono+6-DMAP两种方法激活重构胚,Iono+6-DMAP试验组重构胚的卵裂率和囊胚率高于EH+6-DMAP试验组,但差异不显著。对孤雌胚和核移植胚进行体外培养,培养液改进后,孤雌胚的囊胚率有上升趋势,但差异不显著;胚胎培养液中添加10 ng/mL的EGF提高了重构胚的卵裂率和囊胚率,分别为87.4%和19.3%。
3盘羊供体细胞对异种核移植效率的影响
对传代的细胞进行细胞周期测定,随着汇合度的增加、汇合时间和血清饥饿时间的延长,G0/G1期细胞比例随之增加。
以来自不同个体、不同代次、不同汇合度、不同血清饥饿时间、不同保存方式和不同光滑程度的盘羊成纤维细胞为核供体,以当地绵羊卵母细胞为受体,进行异种重构胚的构建。结果表明:(1)来自3只盘羊耳皮肤成纤维细胞的异种重构胚的卵裂率与囊胚率均无显著差异。(2)采用10代以内的细胞作为核供体,细胞代次不会影响重构胚的发育率。(3)以不同汇合度和汇合不同天数的盘羊成纤维细胞作为供体细胞,重构胚的卵裂率之间没有显著的差异,70%～80%汇合组、90%汇合组和100%汇合1 d组的重构胚的囊胚率显著高于100%汇合2～4 d组重构胚的囊胚率。(4)血清饥饿不同时间的盘羊成纤维细胞,对异种重构胚的发育率没有产生显著的影响。(5)将供体细胞4℃保存2 d,虽降低了卵裂率,但囊胚率没有受到显著影响。(6)选取表面光滑的细胞作为核供体,其融合率显著高于表面粗糙供体细胞组;两试验组重构胚的卵裂率之间没有显著差异,但表面光滑供体细胞组的囊胚率显著高于表面粗糙供体细胞组的囊胚率。因此,利用本试验中的3只盘羊耳成纤维细胞,选择培养到10代以内、70%～80%汇合后的光滑细胞用于构建异种重构胚,能得到较高的早期胚胎发育率。
4不同类型胚胎的体外发育及胚胎移植
利用孤雌激活技术、体外受精技术及体细胞核移植技术分别生产了不同类型的体外胚胎,比较了由不同生产方式引起的遗传物质差异对胚胎体外发育率及囊胚总细胞数的影响。同时,本试验还研究了季节、卵巢保存条件和培养液对PAEs和FC-NTEs发育能力的影响。结果表明:(1)PAEs、FC-NTEs、CC-NTEs、AC-NTEs和IVFEs的卵裂率没有显著差异;PAEs的囊胚率(23.4%)显著高于AC-NTEs(15.9%)组,其它三组差异不显著;FC-NTEs的囊胚细胞数(120)显著多于PAEs的囊胚细胞数(96),与IVFEs的囊胚细胞数(107)没有显著差异。(2)繁殖季节卵巢的PAEs和FC-NTEs的发育率分别显著高于非繁殖季节的发育率。(3)卵巢在14～18℃保存15～17 h,没有影响PAEs的体外发育;却显著降低了FC-NTEs的卵裂率,且没有得到囊胚。(4)SOFaa和CR1aa培养基对PAEs和FC-NTEs的早期发育没有显著影响.(5)进行了一次异种重构胚的移植试验,成熟卵数57枚,核移植53枚,融合率为84.9%,卵裂率为77.8%,将卵裂的35枚胚胎移植到3只受体羊,其中有2只受体羊延迟到90 d后返情,最终未得到妊娠。由此可见,胚胎的体外生产方式和卵巢的来源对早期胚胎的质量和发育率有显著的影响,而SOFaa和CR1aa培养基对胚胎的生长发育影响不大;可以尝试利用异种核移植技术保护濒危动物。
The argali(Ovis ammon) is a conserved and endangered wild species.Because of being over-killed for a long time,the argali(Ovis ammon) have become endangered. Interspecies cloning has shown the great potential to conserve these endangered species.In the present study,we established argali(Ovis ammon) ear fibroblast cell lines by cell culture in vitro,and got higher development in vitro of argali(Ovis ammon)-sheep embryos by optimizing the procedures of nuclear transfer.The argali(Ovis ammon)-sheep embryos were transferred into sheep surrogate mothers,but two of these surrogate sheeps delayed estrus at 90~(th).day and finally no pregnancies were established.
1 Establishment of donor cell lines
We established argali(Ovis ammon) and sheep fibroblast cell lines by tissue piece culture,which were from wild argali(Ovis ammon) car skin and sheep fetus skin in Xinjiang,and cultured and passaged cumulus cells from cumulus-oocyte-complexes by in vitro maturation.The growth curves of argali(Ovis ammon) fibroblasts、sheep fibroblasts and cumulus cells showed that latent phase of argali(Ovis ammon) fibroblasts was longer than that of sheep fibroblasts and cumulus cells.Argali(Ovis ammon) fibroblasts had no obvious plateau phase.The regular fluctuation of growth curve exhibited in vitro growth characteristics of fibroblasts.The fibroblasts subcultured like this could be used as nuclear transfer donors.
In order to establish cryopreservation system for argali(Ovis ammon) fibroblasts,the effect of cryopreservation protocols and cryoprotectants on survival rate of argali(Ovis ammon) fibroblasts after being thawed were studied.Among three cryopreservation protocols,the survival rate of cells was 95.2%by protocol 1 and significantly higher than that by another two protocols.The adhesion rate reached 70%～80%at 18 h after being thawed.The protocol 1 was that cells could be equilibrated at 4℃for 30min,—20℃for 3h,—70℃overnight,and then finally plunged into liquid nitrogen.In protocol 1,cells could stay at—20℃for 3～5h which didn't affect survival rate and growth state after being thawed.The survival and adhesion rate of cells cryopreserved with cryoprotectant 1(50%FBS) were higher than those with cryoprotectant 2(20%FBS).The results imply that cells cryopreserved with 50%FBS cryoprotectant by protocol 1 have higher survival rate after being thawed.
Chromosomal karyotypes of wild argali(Ovis ammon) and sheep in Xinjiang were studied by using the routine analysis method of chromosomal karyotype.The results showed that the number of the chromosomes in argali(Ovis ammon) was 2n= 56.There were 27 pairs of autosomes and 1 pair of sex chromosome.No.1 and No.2 of autosomes were metacentric chromosomes while No.3～No.27 of autosomes were telocentric chromosomes;X chromosome was the largest telocentric chromosome while Y was the smallest metacentric chromosome in all chromosomes.The number of the chromosomes in sheep was 2n=54.There were 26 pairs of autosomes and 1 pair of sex chromosome.Among 26 pairs of autosome,No.1,No.2 and No.3 chromosomes were metacentric chromosomes, while No.4～No.26 chromosomes were telocentric chromosomes;X chromosome was the largest telocentric chromosome while Y chromosome was the smallest metacentric chromosome.
2 Reconstruction of argali(Ovis ammon)-sheep interspecies cloned embryos
The present study was conducted to investigate the effects of storage temperature,time and solution of sheep ovaries on the parthenogenetic developmental competence of oocytes obtained from stored ovaries.The results showed that storage of ovaries at 14～18℃for 15～17h did not affect oocyte maturation(80.9%vs 82.2%) and the potential of oocytes to develop into blastocysts after parthenogenetic activation(21.0%vs 22.0%).The type of storage solutions including modified phosphate-buffered saline(D-PBS) and physiological saline that the ovaries were immersed in had little effect on the results. However,the parthenogenetic oocytes from ovaries stored at 4℃or 30℃for 15～17h had a significantly decreased maturation rate(61.9%vs 19%) and cleavage rate(8.6%vs 9.1%), and did not develop into blastocysts.Thus,sheep ovaries can be stored at 14～18℃for 15～17h without decreasing oocyte maturation competence and the developmental potential of parthenogenetically activated oocytes.
Enucleation of recipient oocytes is critical to nuclear transfer.Blind enucleation is a conventional enucleation method.Chemically assisted enucleation is that the location of sheep oocyte chromosome was displayed with colchicine and enucleated by micromanipulator.In this experiment,it focuses on the influence of the concertration of colchicine handling oocytes and the time of oocytes incubating in colchicine on displaying effect.The effects of blind enucleation and chemically assisted enucleation on enucleation rate were also studied.The results showed that(1) the rates of cytoplast protrusion and enueleation had no difference for ooeytes incubating in colehicine(0.4μg/mL) for 0.5h or 1h.(2) oocytes could be enucleated efficiently after being treated with medium containing 0.2μg/mL or 0.4μg/mL colchicine for 0.5h.(3) maturation for 18～23h didn't affect the rates of cytoplast protrusion and enucleation,and the enucleation rote of oocyes matured for 21～23h was significantly higher by chemically assisted enucleation than blind enucleation. (4) the development rate of reconstructed embryos hadn't been.influenced when oocytes matured for 18～20h were enucleated by blind enucleation and chemically assisted enucleation.It has demonstrated that matured oocytes incubating in colchicine(0.2μg/mL) for 0.5h could not only been enueleated fast and effectively,but didn't affect the early development of reconstituted embryos.
The effects of electrofusion protocol,activation method and embryo culture media on the development of interspecies reconstructed embryos were examined.Results showed that the appropriate electrofusion parameters for interspecies cloned embryos were 121～125 V/mm of pulse intensity,40μs of pulse duration and 2 of pulse number or 126～130 V/mm of pulse intensity,20μs of pulse duration and 2 of pulse number.When those embryos which failed to fusion were refused with the same electrofusion parameters,the development of reconstituted embryos couldn't been influenced and more usable embryos were produced.Parthenogenetic oocytes matured for 25h or 26h resulted in the higher blastocyst rate than that matured for 24h;the higher activation rate was obtained when oocytes were treated with 6—DMAP for 2h;reconstituted embryos activated by Iono+6-DMAP had higher cleavage rate and blastocyst rate than those activated by EH+6-DMAP,but there was no significant difference.The development rate of blastocyst would tend to arise in new culture media though there was no significant difference. Embryo culture media with the addition of 10 ng/mL EGF enhanced the cleavage rate and blastocyst rate of interspecies reconstituted embryos(87.4%,19.3%,respectively).
3 Effects of argali(Ovis ammon) donor cells on interspecies nuclear transfer
We tested the cell cycle stages of argali(Ovis ammon) fibroblasts under different treatment conditions.Results showed that confluency,confluency duration and serum-starvation period had a positive impact on the percentage of G0/G1 cell.
The aim of this study was to investigate the effect of individual,passage number, confluency,serum-starvation period,storage method and surface state of argali(Ovis ammon) fibroblasts on the development of embryos.The embryos were reconstructed by interspecies nuclear transfer using local ovine oocyte cytoplasm.Results showed that(1) there was no difference in the cleavage rate and blastocyst rate among three argali sheeps. (2) the passage number of fibroblast cells within 10 passages didn't affect the development rate of reconstructed embryos.(3) the blastocyst rates,which was produced by 70%～80% confluency,90%confluency and full-confluency for 1d fibroblast cells,were higher than those using full-confluency for 2～4d cells.(4) the serum-starvation period of donor cells didn't affect the development of embryos.(5) the cleavage rate of embryos reconstructed using donor cells stored for 2 days at 4℃was lower than that using donor cells from control group(P＜0.05),but no difference was found in the proportion of blastocysts in total embryos.(6) smooth cells led to a high fusion rate and blastocyst rate.Therefore,The embryos,which were reconstructed by interspecies nuclear transfer using more than 70%～80%confluent smooth fibroblasts within 10 passages from three argali sheeps in Xinjiang,had higher development competence.
4 The in vitro development of ovine embryos produced by different manner and embryo transfer
We conducted the present study to examine the developmental competence of preimplantation ovine embryos in vitro.Parthenogenetic activation(PA),somatic cell nuclear transfer(SCNT) and in vitro fertilization(IVF) were used for ovine PA embryos (PAEs),IVF embryos(IVFEs),ovine fetal fibroblast cell nuclear transfer embryos (FC-NTEs),ovine cumulus cell nuclear transfer embryos(CC-NTEs) and argali(Ovis ammon) fibroblast cell nuclear transfer embryos(AC-NTEs).These embryos were used to study the effects of genetic material differences on the developmental competence in vitro and the total cell number in the blastocyst,which caused by the different embryo-produced manner.Furthermore,effects of season,ovary storage,and embryonic culture media on the developmental potential of the ovine PA embryos(PAEs) and SCNT embryos(NTEs) were also investigated.The results showed:(1) there was no difference in the cleavage rates among PAEs,FC-NTEs,CC-NTEs,AC-NTEs and IVFEs.Blastocyst formation rate of PAEs was significantly higher than that of AC-NTEs(23.4%vs 15.9%),while no significant difference was obtained among blastocyst formation rate of FC-NTEs, CC-NTEs and IVFEs.The total cell number of FC-NT blastocyst was more than that of PA blastocyst(120 vs 96),but had no significant difference with that of IVF blastocyst(120 vs 107).(2) the developmental rates of PAEs and FC-NTEs were significantly higher during estrous seasons than those during anestrous seasons.(3) storaging ovaries at 14～18℃for 15～17 h did not affect the development in vitro of PAEs,but the cleavage rate of FC-NTEs was significantly decreased and no blastocyst was obtained.(4) there was no effect on the preimplantation development of PAEs and FC-NTEs that were cultured in SOFaa and CR1aa media.(5) we had maturated 57 oocytes and got 53 interspecies reconstructed embryos.The fusion rate was 84.9%and the cleavage rate was 77.8%.35 embryos were transferred to 3 surrogate mothers,but two of them delayed estrus at 90~(th) day and finally no pregnancy had been achieved.
引文
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