提高绵羊体细胞核移植效率的研究
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摘要
本研究对绵羊卵母细胞体外成熟、卵母细胞冷冻保存、卵母细胞孤雌激活、绵羊同种和异种体细胞核移植及重构胚胎体外培养等关键技术进行了试验分析,以期提高绵羊体细胞核移植的效率,建立完善的核移植技术体系,为进一步获得体细胞克隆绵羊和异种克隆个体提供技术支撑。本研究内容包括以下几个方面:(1)在绵羊卵母细胞成熟培养液中添加孕酮等某些影响体外成熟的成份,确定绵羊卵母细胞的最佳成熟方案。(2)并通过对比不同冷冻保护剂对卵母细胞的冷冻保存效果,为核移植操作批量提供成熟的卵母细胞。(3)采用胞质注射法,建立精子提取物激活卵母细胞的方法,并且与离子霉素结合6-DMAP激活法和电激活法进行了比较,筛选出适宜激活方法。(4)体外分离培养得到道赛特绵羊和莎能奶山羊的耳皮肤成纤维样细胞,对绵羊皮肤成纤维细胞的生物学特性和不同细胞因子对其生长增殖的影响进行了研究,并对绵羊成纤维细胞和山羊成纤维细胞分别进行体外传代及冻存,为绵羊体细胞核移植和绵羊-山羊种间核移植试验提供了供体细胞。(5)绵羊孤雌激活胚、绵羊重构胚和绵羊-山羊种间重构胚分别置于不同的培养系统中培养,优化绵羊胚胎体外体系,增加胚胎的体外发育潜力。主要研究结果如下:
1.绵羊卵母细胞体外成熟基础液中分别添加5μg/mL FSH+1.0 IU/mL LH+10%发情牛血清(OCS)、5μg/mL FSH+1.0 IU/mL LH+10%胎牛血清(FBS)、0.075 IU/mL HMG+OCS和0.075 IU/mL HMG+10% FBS,结果表明成熟液中添加OCS更有利于卵母细胞的成熟;添加5%～10%的BFF能够提高卵母细胞的成熟率,但20%BFF卵母细胞的体外成熟率下降;培养液中分别添加0.1μg/mL、0.25μg/mL、0.5μg/mL、1.0μg/mL和2.0μg/mL孕酮,结果表明孕酮的最适添加剂量为0.5μg/mL。结果表明绵羊卵母细胞的体外成熟培养液组成为M199+10 mmol/L Hepes+0.38 mmol/L丙酮酸钠+ 25 mmol/L谷氨酰胺+1μg/mL 17β雌二醇(17β-E2)+5μg/mL FSH+1.0 IU/mL LH+10%OCS+0.5μg/mL孕酮时其体外成熟培养效果最好。
2.采用程序冷冻法对处于不同发育时期的绵羊卵母细胞进行冷冻保存,冷冻液Ⅰ～冷冻液Ⅳ对同一时期卵母细胞冷冻解冻后的形态正常率、卵裂率和桑囊胚发育率均无显著影响,而冷冻液Ⅳ体外成熟24 h冷冻解冻后的卵母细胞发育率显著高于GV期和体外成熟10 h的卵母细胞,说明用乙二醇(EG)作为冷冻保护剂,添加0.1 mol/L海藻糖较合适对成熟培养24 h的绵羊卵母细胞进行冷冻保存;-6.5℃植冰时冷冻卵母细胞囊胚发育率显著高于其他组。采用玻璃化冷冻方法对绵羊GV期到体外成熟培养24 h卵母细胞进行冷冻保存试验证实,以玻璃化冷冻液Ⅱ作为冷冻剂,体外成熟培养24 h卵母细胞的卵裂率和囊胚发育率最好,不经过成熟培养的卵母细胞玻璃化冷冻效果最差,认为添加0.1 mol/ L海藻糖来进行卵母细胞的玻璃化冷冻可以起到保护作用,卵母细胞冷冻解冻后可以用作受体胞质进行核移植,重构胚的发育能力受冷冻处理的影响不显著。
3.对绵羊卵母细胞以连续3次120 v/mm的直流脉冲进行电激活获得最佳激活效果,囊胚发育率显著高于脉冲1次和2次(P<0.05);用分别注射不同剂量的精子提取物,对绵羊卵母细胞进行孤雌激活,结果表明最佳注射剂量为向每个卵母细胞注入精子提取物3 pL;分别用电激活法,离子霉素联合6-DMAP法和精子提取物胞质内注射法对绵羊卵母细胞进行激活,结果表明电脉冲的激活效果显著低于后二者的效果,且差异显著,精子提取物和离子霉素联合6-DMAP法的囊胚率差异不显著,均可以用于绵羊卵母细胞的激活。
4.高糖DMEM较适宜于绵羊皮肤成纤维细胞的原代培养和继代培养。以DMEM+50% NCS(new - born calf serum),添加10% DMSO为冻存保护剂,进行冷冻保存的无角道赛特绵羊皮肤成纤维细胞,解冻细胞经体外5~6次传代培养,细胞形态维持在初始状况,保持了良好的遗传特性;添加EGF和胰岛素对绵羊皮肤成纤维细胞的生长均有促生长作用。
5.卵母细胞体外成熟培养时间分别为15~17 h、19~21 h与23~25 h时,随着培养时间的增加成熟率也增加,卵母细胞去核成功率随着成熟时间的延长而下降,试验认为卵母细胞体外成熟19~21 h,既可以保证高的去核成功率,又可以保证卵母细胞较高的成功率,有利于提供充足的卵源;不经血清饥饿处理的供体细胞采用胞质注射法构建克隆胚,其胚胎发育率略高于血清饥饿处理组,但差异不显著。试验认为体外成熟培养19~21 h的卵母细胞可以作为核移植受体细胞,未经血清饥饿处理的成纤维细胞作为供体细胞进行核移植,可以获得较高的重构胚胎发育率。
6.采用胞质内注射法构建绵羊皮肤纤维细胞重组胚,胚胎经离子霉素联合6-DMAP激活后,其细胞融合率、卵裂率和囊胚发育率均高于电融合法,同时结果表明重构胚用离子霉素联合6-DMAP或精子提取物激活后,卵裂率和囊胚发育率相近,其中精子提取物的激活效果优于离子霉素激活法;激活间隔时间研究结果证实,卵母细胞注核后经体外培养3～4 h后再激活,可以获得较高的激活率。
7.用冷冻的山羊成纤维细胞可以成功构建山羊-绵羊异种体细胞核移植胚,种间体细胞核移植胚能够进行正常的早期发育,种间重构胚经离子霉素联合6-DMAP或者胞质注射精子提取物激活后,卵裂率和囊胚发育率差异不显著(P>0.05);用电脉冲融合法构建异种核移植胚胎时,其融合率、卵裂率和囊胚发育率均低于胞质注射法;不同山羊供体细胞进行山羊-绵羊异种核移植胚胎构建时,血清饥饿处理与否对种间核移植囊胚率无显著影响,表明山羊皮肤成纤维细胞可以不经过血清饥饿处理直接进行核移植。
8.联合使用培养液能改善绵羊同种和异种重构胚的发育。即使用CR1aa+BSA培养重构胚3 d,再用SOFaa+5%FBS继续培养,获得的重构胚囊胚发育率较高。在胚胎培养液中添加孕酮可在一定程度上提高重构胚的卵裂率和囊胚率。其中以2.0和4.0μg/mL孕酮组的囊胚率最高,与对照组和其他孕酮浓度组差异显著。发现共培养体系对绵羊同种核移植重构胚的发育有显著的影响,而且在3 d更换1次新的饲养细胞单层对重构胚的发育有明显的促进作用。
In this paper,the maturation, cryopreservation and activation of sheep oocytes in vitro were indicated and analysised the effect of nuclear transfer (NT) and embryo culture methods on the developmental efficiency of homogeneous and heterogeneous reconstructed embryos. The studies included several following researches: (1) different additives such as progesterone added in oocyte maturation culture medium were studied the effect of those additives on the maturation rate of sheep oocytes. (2) different concentration of fucose were optimized on the effect of the sheep oocytes cryopreservation, so enough oocytes can be provided for the sheep NT. (3) the effect of intracytoplasmic injection of sperm extraction on the cleavage rate of the sheep oocytes and the developmental rate of the blastocysts were studied and compared with the traditional chemical activation method using ionomycin and 6-DMAP. (4) sheep and goat ears skin fibroblast cells were cultured in vitro and passaged and the effect of different concentration of insulin and EGF on the proliferation velocity of the sheep fibroblast cells were studied. Then these cells were used for NT manipulation, homogeneous and heterogeneous reconstructed embryos were produced. (5) the effect of different culture media on the developmental capability of parthenogenetic embryos, homogeneous or heterogeneous reconstructed embryos were studied . The results as following:
1.Four groups with different combined additives ( 5μg/mL FSH+1.0 IU/mL LH+10% OCS; 5μg/mL FSH+1.0 IU/mL LH+10%FBS; 0.075 IU/mL HMG+OCS; 0.075 IU/mL HMG+10%FBS) were dissolved into the oocytes maturation media (OM) .This result indicated that OCS was more favorable than FBS for the sheep oocyte maturation. Addition of 5%～10% BFF was useful for the oocytes maturation. However, 20% BFF inhibited the maturation capacity of sheep oocytes When 0.1μg/mL, 0.25μg/mL, 0.5μg/mL, 1.0μg/mL and 2.0μg/mL progesterone were added in OM media ,the results demonstrated addition of 0.5μg/mL progesterone was the optimized concentration for in vitro maturation of sheep oocytes. Thus the followed compositive culture medium was proposed for sheep oocytes maturation in vitro: TCM199+10 mmol/L Hepes+0.38 mmol/L pyruvate sodium+ 25 mmol/L glutamine+1μg/mL 17β-E2+5μg/mL FSH+1.0 IU/mL LH+10%OCS+0.5μg/mL progesterone.
2. The effect of different cytopreservation media and different developmental stage of oocytes on the developmental rates were compared. The cytopreservation media had no significantly impact on the rate of oocyte cleavage and blastocyst,the results indicated that containing EG and 0.1 mol/L fucose in cyropreservation medium was suitable for freezing sheep oocytes cultured for 24 h;The blastocyst rate of reconstructed embryos using donor cells treated with serum starvation or the control was not different significantly. The cyropreserved oocytes could be used as recipients for NT and was not significantly effect on the developmental abilities of the reconstructed embryos.
3. Sheep oocyte with 120 v/mm electric pulse activation for 3 times could get the higher blastocyst rate than the activation rate of 1 times or 2 times (P<0.05). Sperm extraction ( 5 ~6 mg/mL) was injected into sheep cytoplasm to active oocyte , three different doses(3pL、6 pL and 9 pL ) were selected , the results showed the two methods are not different significantly, but both were significantly different with the electric pulse method.
4 . DMEM was suitable for sheep and goat cell culture. We cyropreserved sheep and goat cells by adding 10% DMSO after thawing and culturing 5~6 passages, the cells still maintained the same viability as the primary cells, the study used these cells as nuclear donors for sheep NT and sheep-goat inter-specific NT and obtained a high blastcyst rate. EGF and Insulin were added into DMEM medium for sheep cells culture. EGF and Insulin can both significantly improve the proliferation of sheep cells and can significantly cooperate with each other. But the effect of concentration of Insulin on the cell proliferation didn’t differ with each other significantly.
5. Three groups at different maturation times(15~17 h、19~21 h and 23~25 h) were compared . According to the experiment results, the recommended culture time of oocyte should be 19~21h for the both high rates of maturation and de-nucleation. The blastocyst rate between donor cells treated with serum starvation and the control were no difference significantly.
6. Injecting sheep skin fibroblast cells nucleus into oocyte cytoplasm to reconstruct embryos and using Ionomycin and 6-DMAP to active it, the rate of oocytes fusion are higher than the active fusion results. 3-4 h interval between reconstructed and activate oocytes obtained a higher activation rate.
7. The goat fibroblast cells were used as nucler donors and the ovine oocytes were used as the cell recipients. The activation methods used intracytoplasmic injection of sperm extraction or Ionomycin combined with 6-DMAP were applied to active the presumptive reconstructed embryos. The blastocyst rate between using donor cells treated with serum starvation and the control as nucler donors were no difference significantly.
8. The result showed that the blastocyst rate was improved when ovine homogeneous and heterogeneous reconstructed embyos were cultured in SOFaa +CR1aa(first cultured in CR1aa +BSA,then in SOFaa subsequentially). 2.0 and 4.0μg/mL progesterone were separately added into embryo culture medium,the percentage of blastocysts was higher than control and others concentration group.Co-cultured systems with oviduct epithelial cell or granular cells can improve the development of blastocysts. Changed co-cultured feed layer could significantly improved the development of ovine homogeneous reconstructed embryos.
1.绵羊卵母细胞体外成熟基础液中分别添加5μg/mL FSH+1.0 IU/mL LH+10%发情牛血清(OCS)、5μg/mL FSH+1.0 IU/mL LH+10%胎牛血清(FBS)、0.075 IU/mL HMG+OCS和0.075 IU/mL HMG+10% FBS,结果表明成熟液中添加OCS更有利于卵母细胞的成熟;添加5%～10%的BFF能够提高卵母细胞的成熟率,但20%BFF卵母细胞的体外成熟率下降;培养液中分别添加0.1μg/mL、0.25μg/mL、0.5μg/mL、1.0μg/mL和2.0μg/mL孕酮,结果表明孕酮的最适添加剂量为0.5μg/mL。结果表明绵羊卵母细胞的体外成熟培养液组成为M199+10 mmol/L Hepes+0.38 mmol/L丙酮酸钠+ 25 mmol/L谷氨酰胺+1μg/mL 17β雌二醇(17β-E2)+5μg/mL FSH+1.0 IU/mL LH+10%OCS+0.5μg/mL孕酮时其体外成熟培养效果最好。
2.采用程序冷冻法对处于不同发育时期的绵羊卵母细胞进行冷冻保存,冷冻液Ⅰ～冷冻液Ⅳ对同一时期卵母细胞冷冻解冻后的形态正常率、卵裂率和桑囊胚发育率均无显著影响,而冷冻液Ⅳ体外成熟24 h冷冻解冻后的卵母细胞发育率显著高于GV期和体外成熟10 h的卵母细胞,说明用乙二醇(EG)作为冷冻保护剂,添加0.1 mol/L海藻糖较合适对成熟培养24 h的绵羊卵母细胞进行冷冻保存;-6.5℃植冰时冷冻卵母细胞囊胚发育率显著高于其他组。采用玻璃化冷冻方法对绵羊GV期到体外成熟培养24 h卵母细胞进行冷冻保存试验证实,以玻璃化冷冻液Ⅱ作为冷冻剂,体外成熟培养24 h卵母细胞的卵裂率和囊胚发育率最好,不经过成熟培养的卵母细胞玻璃化冷冻效果最差,认为添加0.1 mol/ L海藻糖来进行卵母细胞的玻璃化冷冻可以起到保护作用,卵母细胞冷冻解冻后可以用作受体胞质进行核移植,重构胚的发育能力受冷冻处理的影响不显著。
3.对绵羊卵母细胞以连续3次120 v/mm的直流脉冲进行电激活获得最佳激活效果,囊胚发育率显著高于脉冲1次和2次(P<0.05);用分别注射不同剂量的精子提取物,对绵羊卵母细胞进行孤雌激活,结果表明最佳注射剂量为向每个卵母细胞注入精子提取物3 pL;分别用电激活法,离子霉素联合6-DMAP法和精子提取物胞质内注射法对绵羊卵母细胞进行激活,结果表明电脉冲的激活效果显著低于后二者的效果,且差异显著,精子提取物和离子霉素联合6-DMAP法的囊胚率差异不显著,均可以用于绵羊卵母细胞的激活。
4.高糖DMEM较适宜于绵羊皮肤成纤维细胞的原代培养和继代培养。以DMEM+50% NCS(new - born calf serum),添加10% DMSO为冻存保护剂,进行冷冻保存的无角道赛特绵羊皮肤成纤维细胞,解冻细胞经体外5~6次传代培养,细胞形态维持在初始状况,保持了良好的遗传特性;添加EGF和胰岛素对绵羊皮肤成纤维细胞的生长均有促生长作用。
5.卵母细胞体外成熟培养时间分别为15~17 h、19~21 h与23~25 h时,随着培养时间的增加成熟率也增加,卵母细胞去核成功率随着成熟时间的延长而下降,试验认为卵母细胞体外成熟19~21 h,既可以保证高的去核成功率,又可以保证卵母细胞较高的成功率,有利于提供充足的卵源;不经血清饥饿处理的供体细胞采用胞质注射法构建克隆胚,其胚胎发育率略高于血清饥饿处理组,但差异不显著。试验认为体外成熟培养19~21 h的卵母细胞可以作为核移植受体细胞,未经血清饥饿处理的成纤维细胞作为供体细胞进行核移植,可以获得较高的重构胚胎发育率。
6.采用胞质内注射法构建绵羊皮肤纤维细胞重组胚,胚胎经离子霉素联合6-DMAP激活后,其细胞融合率、卵裂率和囊胚发育率均高于电融合法,同时结果表明重构胚用离子霉素联合6-DMAP或精子提取物激活后,卵裂率和囊胚发育率相近,其中精子提取物的激活效果优于离子霉素激活法;激活间隔时间研究结果证实,卵母细胞注核后经体外培养3～4 h后再激活,可以获得较高的激活率。
7.用冷冻的山羊成纤维细胞可以成功构建山羊-绵羊异种体细胞核移植胚,种间体细胞核移植胚能够进行正常的早期发育,种间重构胚经离子霉素联合6-DMAP或者胞质注射精子提取物激活后,卵裂率和囊胚发育率差异不显著(P>0.05);用电脉冲融合法构建异种核移植胚胎时,其融合率、卵裂率和囊胚发育率均低于胞质注射法;不同山羊供体细胞进行山羊-绵羊异种核移植胚胎构建时,血清饥饿处理与否对种间核移植囊胚率无显著影响,表明山羊皮肤成纤维细胞可以不经过血清饥饿处理直接进行核移植。
8.联合使用培养液能改善绵羊同种和异种重构胚的发育。即使用CR1aa+BSA培养重构胚3 d,再用SOFaa+5%FBS继续培养,获得的重构胚囊胚发育率较高。在胚胎培养液中添加孕酮可在一定程度上提高重构胚的卵裂率和囊胚率。其中以2.0和4.0μg/mL孕酮组的囊胚率最高,与对照组和其他孕酮浓度组差异显著。发现共培养体系对绵羊同种核移植重构胚的发育有显著的影响,而且在3 d更换1次新的饲养细胞单层对重构胚的发育有明显的促进作用。
In this paper,the maturation, cryopreservation and activation of sheep oocytes in vitro were indicated and analysised the effect of nuclear transfer (NT) and embryo culture methods on the developmental efficiency of homogeneous and heterogeneous reconstructed embryos. The studies included several following researches: (1) different additives such as progesterone added in oocyte maturation culture medium were studied the effect of those additives on the maturation rate of sheep oocytes. (2) different concentration of fucose were optimized on the effect of the sheep oocytes cryopreservation, so enough oocytes can be provided for the sheep NT. (3) the effect of intracytoplasmic injection of sperm extraction on the cleavage rate of the sheep oocytes and the developmental rate of the blastocysts were studied and compared with the traditional chemical activation method using ionomycin and 6-DMAP. (4) sheep and goat ears skin fibroblast cells were cultured in vitro and passaged and the effect of different concentration of insulin and EGF on the proliferation velocity of the sheep fibroblast cells were studied. Then these cells were used for NT manipulation, homogeneous and heterogeneous reconstructed embryos were produced. (5) the effect of different culture media on the developmental capability of parthenogenetic embryos, homogeneous or heterogeneous reconstructed embryos were studied . The results as following:
1.Four groups with different combined additives ( 5μg/mL FSH+1.0 IU/mL LH+10% OCS; 5μg/mL FSH+1.0 IU/mL LH+10%FBS; 0.075 IU/mL HMG+OCS; 0.075 IU/mL HMG+10%FBS) were dissolved into the oocytes maturation media (OM) .This result indicated that OCS was more favorable than FBS for the sheep oocyte maturation. Addition of 5%～10% BFF was useful for the oocytes maturation. However, 20% BFF inhibited the maturation capacity of sheep oocytes When 0.1μg/mL, 0.25μg/mL, 0.5μg/mL, 1.0μg/mL and 2.0μg/mL progesterone were added in OM media ,the results demonstrated addition of 0.5μg/mL progesterone was the optimized concentration for in vitro maturation of sheep oocytes. Thus the followed compositive culture medium was proposed for sheep oocytes maturation in vitro: TCM199+10 mmol/L Hepes+0.38 mmol/L pyruvate sodium+ 25 mmol/L glutamine+1μg/mL 17β-E2+5μg/mL FSH+1.0 IU/mL LH+10%OCS+0.5μg/mL progesterone.
2. The effect of different cytopreservation media and different developmental stage of oocytes on the developmental rates were compared. The cytopreservation media had no significantly impact on the rate of oocyte cleavage and blastocyst,the results indicated that containing EG and 0.1 mol/L fucose in cyropreservation medium was suitable for freezing sheep oocytes cultured for 24 h;The blastocyst rate of reconstructed embryos using donor cells treated with serum starvation or the control was not different significantly. The cyropreserved oocytes could be used as recipients for NT and was not significantly effect on the developmental abilities of the reconstructed embryos.
3. Sheep oocyte with 120 v/mm electric pulse activation for 3 times could get the higher blastocyst rate than the activation rate of 1 times or 2 times (P<0.05). Sperm extraction ( 5 ~6 mg/mL) was injected into sheep cytoplasm to active oocyte , three different doses(3pL、6 pL and 9 pL ) were selected , the results showed the two methods are not different significantly, but both were significantly different with the electric pulse method.
4 . DMEM was suitable for sheep and goat cell culture. We cyropreserved sheep and goat cells by adding 10% DMSO after thawing and culturing 5~6 passages, the cells still maintained the same viability as the primary cells, the study used these cells as nuclear donors for sheep NT and sheep-goat inter-specific NT and obtained a high blastcyst rate. EGF and Insulin were added into DMEM medium for sheep cells culture. EGF and Insulin can both significantly improve the proliferation of sheep cells and can significantly cooperate with each other. But the effect of concentration of Insulin on the cell proliferation didn’t differ with each other significantly.
5. Three groups at different maturation times(15~17 h、19~21 h and 23~25 h) were compared . According to the experiment results, the recommended culture time of oocyte should be 19~21h for the both high rates of maturation and de-nucleation. The blastocyst rate between donor cells treated with serum starvation and the control were no difference significantly.
6. Injecting sheep skin fibroblast cells nucleus into oocyte cytoplasm to reconstruct embryos and using Ionomycin and 6-DMAP to active it, the rate of oocytes fusion are higher than the active fusion results. 3-4 h interval between reconstructed and activate oocytes obtained a higher activation rate.
7. The goat fibroblast cells were used as nucler donors and the ovine oocytes were used as the cell recipients. The activation methods used intracytoplasmic injection of sperm extraction or Ionomycin combined with 6-DMAP were applied to active the presumptive reconstructed embryos. The blastocyst rate between using donor cells treated with serum starvation and the control as nucler donors were no difference significantly.
8. The result showed that the blastocyst rate was improved when ovine homogeneous and heterogeneous reconstructed embyos were cultured in SOFaa +CR1aa(first cultured in CR1aa +BSA,then in SOFaa subsequentially). 2.0 and 4.0μg/mL progesterone were separately added into embryo culture medium,the percentage of blastocysts was higher than control and others concentration group.Co-cultured systems with oviduct epithelial cell or granular cells can improve the development of blastocysts. Changed co-cultured feed layer could significantly improved the development of ovine homogeneous reconstructed embryos.
引文
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