牛体细胞克隆中供体和受体细胞制备及重构胚构建的影响因素研究
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摘要
体细胞克隆技术是哺乳动物胚胎工程的组成部分，它不仅有利于缩短动物繁殖育种时间和提高优良种畜的利用率，也有利于精卵受精机制的研究。在哺乳动物体细胞克隆研究中，从受体卵母细胞培养、供体细胞制备、受体细胞去核、重构胚构建、重构胚激活、重构胚培养、胚胎移植到胎儿出生，每个技术环节都会影响克隆的效率。在这些环节中，供体细胞和受体卵母细胞制备以及重构胚构建尤为关键。本试验进行了牛体细胞克隆中供体和受体细胞生产及重构胚构建的影响因素研究。研究了牛卵巢颗粒细胞和输卵管上皮细胞制备过程中细胞培养代数、血清饥饿浓度和饥饿时间对细胞周期和凋亡的影响，同时研究了血清饥饿诱导细胞产生凋亡的机理和抗氧化剂对细胞凋亡的抑制；通过提前去除培养液中的微量元素铁和铜，得到不含微量元素铁和铜的培养液，然后在培养液中分别添加定量的微量元素铁和铜，通过体外受精技术研究微量元素铁和铜对牛卵母细胞体外成熟和早期胚胎体外发育的影响；研究了低渗液处理供体颗粒细胞对胞质直接注射克隆研究中重构胚构建和随后重构胚发育的影响。
1．研究了体细胞克隆中供体细胞制备过程中细胞培养代数、血清饥饿浓度和饥饿时间对体细胞周期的影响。体细胞为牛卵巢颗粒细胞和输卵管上皮细胞，细胞以碘化丙啶染色法处理，用流式细胞仪检测细胞周期。细胞传代培养至第2代、10代和25代，在0.5％和0.05％的血清饥饿浓度下诱导处理2d、3d、4d和5d。实验结果如下：第2代、10代和25代的细胞在G0／G1期的比例没有显著差异(P＞0.05)。培养液中添加0.5％FCS和0.05％FCS较对照组(10％FCS)显著提高了细胞G0／G1期的比例(P＜0.05)，0.5％FCS和0.05％FCS实验组获得了相近的细胞G0／G1期比例(P＞0.05)。血清饥饿至第5d的细胞较第2d的细胞有高的G0／G1期的比例(P＜0.05)。从结果得知体外培养至不同代的牛卵巢颗粒细胞和输卵管上皮细胞对G0／G1期的比例没有影响，血清饥饿和延长血清饥饿时间能够有效诱导体细胞进入G0／G1期。
2．研究了克隆的供体细胞制备过程中细胞培养代数、血清饥饿和血清饥饿时间对体细胞凋亡的影响，同时研究了血清饥饿诱导细胞产生凋亡的机理。体细胞为牛卵巢颗粒细胞和输卵管上皮细胞，细胞以Annexin V／FITC双染色法染色，用流式细胞仪检测细胞凋亡。细胞传代培养至2代、10代和25代，在0.5％和0.05％的FCS饥饿浓度下诱导2d、3d、4d和5d。Caspase抑制剂z-VAD-fmk用来检测血清饥饿诱导细胞产生的凋亡是否由caspase介导。实验结果如下：第2代和第10代的细胞在凋亡率上差异不显著(P＞0.05)，第25代的细胞较第2代和第10代的细胞有高的凋亡率(P＜0.05)。0.5％FCS和0.05％的FCS处理组显著提高了细胞凋亡率(P＜0.05)，而且0.05％FCS较0.5％FCS有更高的细胞凋亡率(P＜0.05)。血清饥饿处理4d和5d的细胞显著提高了细胞的凋亡率(P＜0.05)。培养液中添加z-VAD-fmk能够显著降低0.5％和0.05％FCS诱导细胞产生的细胞凋亡率(P＜0.05)，但对10％FCS实验组的细胞差异不显著(P＞0.05)。从结果得知牛颗粒细胞和输卵管上皮细胞体外长期培养可提高细胞凋亡率，降低血清饥饿的浓度和延长血清饥饿的时间均可造成细胞高水平的凋亡。血清饥饿诱导牛颗粒细胞和输卵管上皮细胞产生凋亡的过程包括caspase的激活。
3．研究了不同抗氧化剂对在体细胞血清饥饿诱导过程中产生凋亡的影响。体细胞为牛卵巢颗粒细胞和牛输卵管上皮细胞，细胞以AnnexinV／FITC双染色法染色，用流式细胞仪检测细胞凋亡。10％FCS培养的细胞和0.5％FCS血清饥饿诱导处理的第5代细胞在添加抗氧化剂维生素E、硒和谷胱苷肽后培养2d，然后检测细胞凋亡率。实验结果如下：添加有维生素E、硒和谷胺酰胺的10％FCS培养的牛卵巢颗粒细胞较对照组降低了细胞凋亡率，但差异不显著(P＞0.05)。在0.5％FCS饥饿诱导处理的颗粒细胞实验组，对照组的细胞凋亡率显著高于维生素E、硒和谷胺酰胺实验组(P＜0.05)。在10％FCS培养的牛输卵管上皮细胞实验组，抗氧化剂处理组的细胞凋亡率低于实验对照组但差异不显著(P＞0.05)。在0.5％FCS饥饿诱导的牛输卵管上皮细胞实验组，维生素E、硒和谷胺酰胺处理组之间的细胞凋亡率差异不显著(P＞0.05)，但较对照组显著降低了细胞的凋亡率(P＜0.05)。这些结果显示通过在培养过程中分别添加抗氧化剂维生素E、硒和谷胱苷肽能够有效抑制因血清饥饿诱导而产生的细胞凋亡，而抗氧化剂不能抑制10％FCS实验组细胞的凋亡。
4．研究铁和铜对牛卵母细胞体外成熟、早期胚胎生长发育以及囊胚期细胞凋亡的影响。培养液中铁的浓度为：0(对照组)，0.45mg／L，0.81mg／L，1.96mg／L和3.26mg／L；铜的浓度为：0mg／L(对照组)，0.093mg／L，0.27mg／L，0.46mg／L和0.68mg／L。在卵母细胞成熟培养的22h、受精卵培养的48h、96h、144h和192h检测培养液中铁浓度(1.96mg／L)和铜浓度(0.46mg／L)的变化。在实验结果中，铁的不同添加组在卵母细胞成熟和卵裂率方面与对照组差异不显著(p＞0.05)，但是铁显著提高了8细胞胚胎、桑椹胚和囊胚的发育率(p＜0.05)，1.96mg／L的铁浓度显著提高了囊胚率(p＜0.05)。铜和铁对卵母细胞成熟和卵裂有相似的影响，0.46mg／L和0.68mg／L的铜显著提高了桑椹胚和囊胚的发育率(p＜0.05)。和对照组相比，铁和铜显著降低了囊胚期细胞的凋亡率(p＜0.05)。在卵母细胞体外成熟的22h和受精卵培养的48h，培养中铁浓度分别下降了3.6％和9.2％，铜浓度分别下降了6.5％和10.9％。在受精卵培养的96h，144h和192h，培养液中铁浓度分别下降21.4％，25.5％和27.0％，铜浓度分别下降了23.9％，28.3％和30.4％。本试验结果说明：培养液中的铁和铜对早期胚胎培养至8细胞、桑椹胚和囊胚有重要的作用；长期的缺铁或缺铜造成囊胚期细胞凋亡率的上升；在早期胚胎培养至8细胞后，早期胚胎对微量元素铁和铜需求的主体可能由细胞本身转向培养液。
5．研究了0.075 M KCl的低渗处理液在胞质直接注射的克隆研究中对供体细胞质膜破裂的影响，通过判定随后重构胚的形成、重构胚后期发育和囊胚期细胞的凋亡来评价低渗处理液对供体细胞破膜处理的影响。根据0.075 M KCl低渗液对供体颗粒细胞处理的不同时间，将用于克隆的去核卵母细胞分为五组：0s(对照组)，30s，60s，90s和180s。细胞核荧光染色用Hoechst 33342。实验结果如下：0.075 M KCl低渗液对供体颗粒细胞的不同处理时间较对照组显著提高了重构胚的形成，但180 s的实验处理组显著抑制了重构胚发育至囊胚的比例(P＜0.05)，而且180s实验处理组的囊胚期细胞显示出典型的凋亡特征。这些结果显示：在胞质直接注射的克隆研究中，可以通过低渗液处理供体细胞促使供体细胞质膜的破裂来提高重构胚的构建效率，但对供体细胞长时间的低渗处理降低了重构胚发育后期囊胚的发育率，而且提高了囊胚期细胞的凋亡率。
Somatic cell nuclear transfer is a major content of mammal embryo engineering, and it is useful not only for shortening the time of animal breed and increasing the availability of good species but also for the study of fertilization mechanism. There are many steps in the procedure of mammal somatic cell nuclear transfer from recipient oocyte culture, donor cell preparation, nuclear transfer, reconstructed embryo formation, reconstructed embryo activation, reconstructed embryo culture, embryo transfer to foetus birth, and each of these steps influence the efficiency of somatic cell nuclear transfer. In these steps, donor cell preparation, recipient oocyte culture and reconstructed embryo formation play important role. In our study, we investigated several factors which affected donor cell preparation, recipient oocyte culture and reconstructed embryo formation in bovine nuclear transfer. We investigated the effects of culture passages, serum starvation concentration and serum starvation time on cell cycle and apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelia cells, and we also studied the mechanism of apoptosis origined from serum starvation and the inhibit effect of different antioxidant for their apoptosis. We added various concentrations of iron or copper to culture media from which we had previously removed the respective element in order to examine the effects of iron and copper on bovine oocyte maturation and preimplantation embryo development in vitro. Furthermore, our study determined lower osmotic pressure duration optimal for breaking granulosa cell membrane, and to determine its effects on reconstructed embryos formation and reconstructed embryos developmental potential in bovine nuclear transfer by intraplasmic injection.
1. Investigated the effects of culture time, serum starvation and starvation time on cell cycle of in vitro cultured bovine granulosa cells and oviduct epithelium cells. Cell cycleanalysis of propidium iodide staining was performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5% and 0.05% FCS seum starvation for 2 d, 3 d, 4 d, and 5 d. The results were as follows: bovine granulosa cells and oviduct epithelium cells of P 2, P 10 and P 25 have no significant differences in the percentages of G0/G1 phase (P＞0.05). 0.5% and 0.05% FCS in culture medium significantly increased the percentages of G0/G1 phase than their control(10% FCS)(P＜0.05), and there were no significant differences between 0.5% FCS and 0.05% FCS (P＞0.05). The cells cultured for 5 d at 0.5% FCS starvation had significant higher percentages of G0/G1 phase than the cells cultured for 2 d at 0.5% FCS starvation. In concluded that culture time of bovine granulosa cells and oviduct epithelium cells in vitro has no effect on the percentages of G0/G1 phase, and serum starvation and prolonged serum starvation time synchronized the cell cycle effectively.
2. Investigated the effects of culture time, serum starvation and serum starvation time on apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelium cells and investigated its possible mechanisms. Cell apoptosis detection of Annexin V/FITC double staining were performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5%FCS and 0.05% FCS seum starvation for 2 d, 3 d, 4 d and 5 d. Caspase inhibitor z-VAD-fmk was used to determine whether serum starvation-induced apoptosis was mediated by caspase activation. The results were as follows: there were no significant differences in apoptosis rates of granulosa cells between P 2 and P 10, and the cells of P 25 had significantly higher apoptosis rates than the cells of P 2 and P 10. Apoptosis rates of 0.5%FCS and 0.05%FCS serum starvation were significant differences and significant greater than serum-fed treatment (10% FCS). The cells cultured for 4 d and 5 d at 0.5% FCS starvation had significantly higher apoptosis rates than the cells cultured for 2 d and 3 d. Culture medium with caspase inhibitor z-VAD-fmk significantly decreased the cells apoptosis rates of 0.5% and 0.05% FCS starvation than culture medium without z-VAD-fmk, and they were no significant differences in the cells apoptosis rates of 10% FCS between z-VAD-fmk and the control. In concluded that long-term curture in vitro, serum starvation and prolonged serum starvation time resulted in high apoptosis rate of bovine granulosa cells and oviduct epithelium cells, and serum starvation-induced apoptosis has the involvement of caspases.
3. Investigated the effects of different antioxidant on inhibit of apoptosis origined from serum starvation, and somatic cells were bovine granulosa cells and oviduct epithelium cells. Cell apoptosis detection of Annexin V/FITC double staining was performed by flow cytometric assay. Cells of P 5 were detected in 2 d culture in 10% and 0.5% FCS culture medium with antioxidant vitamin E, selenium and tathion. The results were as follows: 10% FCS treatments with antioxidant vitamin E, selenium and tathion decreased apoptosis rates of granulosa cells compared with their control, but there were no significant differences. However, 0.5% FCS treatments with antioxidant vitamin E, selenium and tathion significantly decreased apoptosis rates of granulosa cells compared with their control. 10% FCS treatments with antioxidant decreased apoptosis rates of oviduct epithelium cells compared with their control, but there were no significant differences, and 0.5% FCS treatments with antioxidant significantly decreased apoptosis rates of the cells compared with their control. In concluded that bovine granulosa cells and oviduct epithelium cells apoptosis induced by serum starvation could be active suppression through adding antioxidant vitamin E, selenium and tathion in culture medium, and antioxidant vitamin E, selenium and tathion in culture medium could not inhibit apoptosis of the cells cultured in 10% FCS culture medium.
4. This study was on the effects of iron and copper on bovine oocytes maturation, preimplantational embryos development and apoptosis in blastocysts. Iron concentrations in culture medium were 0 mg/L(control), 0.45mg/L, 0.81mg/L, 1.96mg/Land 3.26mg/L, and copper were 0mg/L(control), 0.093mg/L, 0.27mg/L, 0.46mg/Land 0.68mg/L. The variance of iron concentration (1.96mg/L) and copper concentration (0.46mg/L) in culture medium were mensurated at 22h of oocytes maturation and at 48h, 96h, 144h and 192h of zygotes culture. The results were as follows: there were no significant differences in oocytes maturation and cleavage between iron and control, but iron had higher (p＜0.05) rate of 8-cell embryos, morulae and blastocysts than the control, and 1.96mg/Lof iron increased blastocysts rate (p＜0.05). The effects of copper on oocytes maturation and cleavage was similar to iron, and 0.46 and 0.68mg/Lof copper increased the rate of morulae and blastocysts (p＜0.05). Iron or copper had a significant decrease in apoptotic blastomeres than the control (p＜0.05). At 22h of oocytes maturation and 48h of zygotes culture, decreased percentage of iron concentrations were 3.6% and 9.2%, respectively, and that of copper were 6.5 and 10.9%, respectively. At 96h, 144h and 192h of zygotes culture, decreased percentage of iron concentrations were 21.4%, 25.5% and 27.0%, respectively, and that of copper were 23.9%, 28.3% and 30.4%, respectively. In conclusion: iron or copper played an important role in the success of culture of 8-cell embryos, morulae and blastocysts, and long-term lack of iron or copper increased apoptotic blastomeres. There might has a transition of primary demand for trace element iron or copper utilized by zygotes from cytoplast to culture medium after 8-cell of in vitro zygotes culture.
5. Determined lower osmotic pressure (0.075 M KCl) duration optimal for breaking granulosa cell membrane, and determined its effects on reconstructed embryos formation, reconstructed embryos developmental potential and cells apoptosis of blastocysts in bovine nuclear transfer by intracytoplasmic injection. Enucleated bovine oocytes were divided into five groups according to duration of 0.075 M KCl to granulosa cells: Os (control), 30s, 60s, 90s and 180s. Typical apoptotic nuclei was shown by staining of granulosa cells with DNA-binding fluorochrome Hoechst 33342. Different duration of 0.075 M KCl significantly increased the formation of reconstructed embryos than control. The treatment of 180s had significantly lower rate of blastocysts than other groups, and nuclei of blastocysts from 180s duration of 0.075 M KCl showed typical apoptosis morphology. These results suggested that lower osmotic pressure to bovine granulosa cells accelerated the formation of reconstructed embryos in nuclear transfer of intracytoplasmic injection, and long duration of lower osmotic pressure resulted in low blastocysts rate and high cells apoptosis rate of blastocysts.
1．研究了体细胞克隆中供体细胞制备过程中细胞培养代数、血清饥饿浓度和饥饿时间对体细胞周期的影响。体细胞为牛卵巢颗粒细胞和输卵管上皮细胞，细胞以碘化丙啶染色法处理，用流式细胞仪检测细胞周期。细胞传代培养至第2代、10代和25代，在0.5％和0.05％的血清饥饿浓度下诱导处理2d、3d、4d和5d。实验结果如下：第2代、10代和25代的细胞在G0／G1期的比例没有显著差异(P＞0.05)。培养液中添加0.5％FCS和0.05％FCS较对照组(10％FCS)显著提高了细胞G0／G1期的比例(P＜0.05)，0.5％FCS和0.05％FCS实验组获得了相近的细胞G0／G1期比例(P＞0.05)。血清饥饿至第5d的细胞较第2d的细胞有高的G0／G1期的比例(P＜0.05)。从结果得知体外培养至不同代的牛卵巢颗粒细胞和输卵管上皮细胞对G0／G1期的比例没有影响，血清饥饿和延长血清饥饿时间能够有效诱导体细胞进入G0／G1期。
2．研究了克隆的供体细胞制备过程中细胞培养代数、血清饥饿和血清饥饿时间对体细胞凋亡的影响，同时研究了血清饥饿诱导细胞产生凋亡的机理。体细胞为牛卵巢颗粒细胞和输卵管上皮细胞，细胞以Annexin V／FITC双染色法染色，用流式细胞仪检测细胞凋亡。细胞传代培养至2代、10代和25代，在0.5％和0.05％的FCS饥饿浓度下诱导2d、3d、4d和5d。Caspase抑制剂z-VAD-fmk用来检测血清饥饿诱导细胞产生的凋亡是否由caspase介导。实验结果如下：第2代和第10代的细胞在凋亡率上差异不显著(P＞0.05)，第25代的细胞较第2代和第10代的细胞有高的凋亡率(P＜0.05)。0.5％FCS和0.05％的FCS处理组显著提高了细胞凋亡率(P＜0.05)，而且0.05％FCS较0.5％FCS有更高的细胞凋亡率(P＜0.05)。血清饥饿处理4d和5d的细胞显著提高了细胞的凋亡率(P＜0.05)。培养液中添加z-VAD-fmk能够显著降低0.5％和0.05％FCS诱导细胞产生的细胞凋亡率(P＜0.05)，但对10％FCS实验组的细胞差异不显著(P＞0.05)。从结果得知牛颗粒细胞和输卵管上皮细胞体外长期培养可提高细胞凋亡率，降低血清饥饿的浓度和延长血清饥饿的时间均可造成细胞高水平的凋亡。血清饥饿诱导牛颗粒细胞和输卵管上皮细胞产生凋亡的过程包括caspase的激活。
3．研究了不同抗氧化剂对在体细胞血清饥饿诱导过程中产生凋亡的影响。体细胞为牛卵巢颗粒细胞和牛输卵管上皮细胞，细胞以AnnexinV／FITC双染色法染色，用流式细胞仪检测细胞凋亡。10％FCS培养的细胞和0.5％FCS血清饥饿诱导处理的第5代细胞在添加抗氧化剂维生素E、硒和谷胱苷肽后培养2d，然后检测细胞凋亡率。实验结果如下：添加有维生素E、硒和谷胺酰胺的10％FCS培养的牛卵巢颗粒细胞较对照组降低了细胞凋亡率，但差异不显著(P＞0.05)。在0.5％FCS饥饿诱导处理的颗粒细胞实验组，对照组的细胞凋亡率显著高于维生素E、硒和谷胺酰胺实验组(P＜0.05)。在10％FCS培养的牛输卵管上皮细胞实验组，抗氧化剂处理组的细胞凋亡率低于实验对照组但差异不显著(P＞0.05)。在0.5％FCS饥饿诱导的牛输卵管上皮细胞实验组，维生素E、硒和谷胺酰胺处理组之间的细胞凋亡率差异不显著(P＞0.05)，但较对照组显著降低了细胞的凋亡率(P＜0.05)。这些结果显示通过在培养过程中分别添加抗氧化剂维生素E、硒和谷胱苷肽能够有效抑制因血清饥饿诱导而产生的细胞凋亡，而抗氧化剂不能抑制10％FCS实验组细胞的凋亡。
4．研究铁和铜对牛卵母细胞体外成熟、早期胚胎生长发育以及囊胚期细胞凋亡的影响。培养液中铁的浓度为：0(对照组)，0.45mg／L，0.81mg／L，1.96mg／L和3.26mg／L；铜的浓度为：0mg／L(对照组)，0.093mg／L，0.27mg／L，0.46mg／L和0.68mg／L。在卵母细胞成熟培养的22h、受精卵培养的48h、96h、144h和192h检测培养液中铁浓度(1.96mg／L)和铜浓度(0.46mg／L)的变化。在实验结果中，铁的不同添加组在卵母细胞成熟和卵裂率方面与对照组差异不显著(p＞0.05)，但是铁显著提高了8细胞胚胎、桑椹胚和囊胚的发育率(p＜0.05)，1.96mg／L的铁浓度显著提高了囊胚率(p＜0.05)。铜和铁对卵母细胞成熟和卵裂有相似的影响，0.46mg／L和0.68mg／L的铜显著提高了桑椹胚和囊胚的发育率(p＜0.05)。和对照组相比，铁和铜显著降低了囊胚期细胞的凋亡率(p＜0.05)。在卵母细胞体外成熟的22h和受精卵培养的48h，培养中铁浓度分别下降了3.6％和9.2％，铜浓度分别下降了6.5％和10.9％。在受精卵培养的96h，144h和192h，培养液中铁浓度分别下降21.4％，25.5％和27.0％，铜浓度分别下降了23.9％，28.3％和30.4％。本试验结果说明：培养液中的铁和铜对早期胚胎培养至8细胞、桑椹胚和囊胚有重要的作用；长期的缺铁或缺铜造成囊胚期细胞凋亡率的上升；在早期胚胎培养至8细胞后，早期胚胎对微量元素铁和铜需求的主体可能由细胞本身转向培养液。
5．研究了0.075 M KCl的低渗处理液在胞质直接注射的克隆研究中对供体细胞质膜破裂的影响，通过判定随后重构胚的形成、重构胚后期发育和囊胚期细胞的凋亡来评价低渗处理液对供体细胞破膜处理的影响。根据0.075 M KCl低渗液对供体颗粒细胞处理的不同时间，将用于克隆的去核卵母细胞分为五组：0s(对照组)，30s，60s，90s和180s。细胞核荧光染色用Hoechst 33342。实验结果如下：0.075 M KCl低渗液对供体颗粒细胞的不同处理时间较对照组显著提高了重构胚的形成，但180 s的实验处理组显著抑制了重构胚发育至囊胚的比例(P＜0.05)，而且180s实验处理组的囊胚期细胞显示出典型的凋亡特征。这些结果显示：在胞质直接注射的克隆研究中，可以通过低渗液处理供体细胞促使供体细胞质膜的破裂来提高重构胚的构建效率，但对供体细胞长时间的低渗处理降低了重构胚发育后期囊胚的发育率，而且提高了囊胚期细胞的凋亡率。
Somatic cell nuclear transfer is a major content of mammal embryo engineering, and it is useful not only for shortening the time of animal breed and increasing the availability of good species but also for the study of fertilization mechanism. There are many steps in the procedure of mammal somatic cell nuclear transfer from recipient oocyte culture, donor cell preparation, nuclear transfer, reconstructed embryo formation, reconstructed embryo activation, reconstructed embryo culture, embryo transfer to foetus birth, and each of these steps influence the efficiency of somatic cell nuclear transfer. In these steps, donor cell preparation, recipient oocyte culture and reconstructed embryo formation play important role. In our study, we investigated several factors which affected donor cell preparation, recipient oocyte culture and reconstructed embryo formation in bovine nuclear transfer. We investigated the effects of culture passages, serum starvation concentration and serum starvation time on cell cycle and apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelia cells, and we also studied the mechanism of apoptosis origined from serum starvation and the inhibit effect of different antioxidant for their apoptosis. We added various concentrations of iron or copper to culture media from which we had previously removed the respective element in order to examine the effects of iron and copper on bovine oocyte maturation and preimplantation embryo development in vitro. Furthermore, our study determined lower osmotic pressure duration optimal for breaking granulosa cell membrane, and to determine its effects on reconstructed embryos formation and reconstructed embryos developmental potential in bovine nuclear transfer by intraplasmic injection.
1. Investigated the effects of culture time, serum starvation and starvation time on cell cycle of in vitro cultured bovine granulosa cells and oviduct epithelium cells. Cell cycleanalysis of propidium iodide staining was performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5% and 0.05% FCS seum starvation for 2 d, 3 d, 4 d, and 5 d. The results were as follows: bovine granulosa cells and oviduct epithelium cells of P 2, P 10 and P 25 have no significant differences in the percentages of G0/G1 phase (P＞0.05). 0.5% and 0.05% FCS in culture medium significantly increased the percentages of G0/G1 phase than their control(10% FCS)(P＜0.05), and there were no significant differences between 0.5% FCS and 0.05% FCS (P＞0.05). The cells cultured for 5 d at 0.5% FCS starvation had significant higher percentages of G0/G1 phase than the cells cultured for 2 d at 0.5% FCS starvation. In concluded that culture time of bovine granulosa cells and oviduct epithelium cells in vitro has no effect on the percentages of G0/G1 phase, and serum starvation and prolonged serum starvation time synchronized the cell cycle effectively.
2. Investigated the effects of culture time, serum starvation and serum starvation time on apoptosis of in vitro cultured bovine granulosa cells and oviduct epithelium cells and investigated its possible mechanisms. Cell apoptosis detection of Annexin V/FITC double staining were performed by flow cytometric assay. Bovine granulosa cells and oviduct epithelium cells were cultured to P 2, P 10 and P 25, and then the cells were cultured in 0.5%FCS and 0.05% FCS seum starvation for 2 d, 3 d, 4 d and 5 d. Caspase inhibitor z-VAD-fmk was used to determine whether serum starvation-induced apoptosis was mediated by caspase activation. The results were as follows: there were no significant differences in apoptosis rates of granulosa cells between P 2 and P 10, and the cells of P 25 had significantly higher apoptosis rates than the cells of P 2 and P 10. Apoptosis rates of 0.5%FCS and 0.05%FCS serum starvation were significant differences and significant greater than serum-fed treatment (10% FCS). The cells cultured for 4 d and 5 d at 0.5% FCS starvation had significantly higher apoptosis rates than the cells cultured for 2 d and 3 d. Culture medium with caspase inhibitor z-VAD-fmk significantly decreased the cells apoptosis rates of 0.5% and 0.05% FCS starvation than culture medium without z-VAD-fmk, and they were no significant differences in the cells apoptosis rates of 10% FCS between z-VAD-fmk and the control. In concluded that long-term curture in vitro, serum starvation and prolonged serum starvation time resulted in high apoptosis rate of bovine granulosa cells and oviduct epithelium cells, and serum starvation-induced apoptosis has the involvement of caspases.
3. Investigated the effects of different antioxidant on inhibit of apoptosis origined from serum starvation, and somatic cells were bovine granulosa cells and oviduct epithelium cells. Cell apoptosis detection of Annexin V/FITC double staining was performed by flow cytometric assay. Cells of P 5 were detected in 2 d culture in 10% and 0.5% FCS culture medium with antioxidant vitamin E, selenium and tathion. The results were as follows: 10% FCS treatments with antioxidant vitamin E, selenium and tathion decreased apoptosis rates of granulosa cells compared with their control, but there were no significant differences. However, 0.5% FCS treatments with antioxidant vitamin E, selenium and tathion significantly decreased apoptosis rates of granulosa cells compared with their control. 10% FCS treatments with antioxidant decreased apoptosis rates of oviduct epithelium cells compared with their control, but there were no significant differences, and 0.5% FCS treatments with antioxidant significantly decreased apoptosis rates of the cells compared with their control. In concluded that bovine granulosa cells and oviduct epithelium cells apoptosis induced by serum starvation could be active suppression through adding antioxidant vitamin E, selenium and tathion in culture medium, and antioxidant vitamin E, selenium and tathion in culture medium could not inhibit apoptosis of the cells cultured in 10% FCS culture medium.
4. This study was on the effects of iron and copper on bovine oocytes maturation, preimplantational embryos development and apoptosis in blastocysts. Iron concentrations in culture medium were 0 mg/L(control), 0.45mg/L, 0.81mg/L, 1.96mg/Land 3.26mg/L, and copper were 0mg/L(control), 0.093mg/L, 0.27mg/L, 0.46mg/Land 0.68mg/L. The variance of iron concentration (1.96mg/L) and copper concentration (0.46mg/L) in culture medium were mensurated at 22h of oocytes maturation and at 48h, 96h, 144h and 192h of zygotes culture. The results were as follows: there were no significant differences in oocytes maturation and cleavage between iron and control, but iron had higher (p＜0.05) rate of 8-cell embryos, morulae and blastocysts than the control, and 1.96mg/Lof iron increased blastocysts rate (p＜0.05). The effects of copper on oocytes maturation and cleavage was similar to iron, and 0.46 and 0.68mg/Lof copper increased the rate of morulae and blastocysts (p＜0.05). Iron or copper had a significant decrease in apoptotic blastomeres than the control (p＜0.05). At 22h of oocytes maturation and 48h of zygotes culture, decreased percentage of iron concentrations were 3.6% and 9.2%, respectively, and that of copper were 6.5 and 10.9%, respectively. At 96h, 144h and 192h of zygotes culture, decreased percentage of iron concentrations were 21.4%, 25.5% and 27.0%, respectively, and that of copper were 23.9%, 28.3% and 30.4%, respectively. In conclusion: iron or copper played an important role in the success of culture of 8-cell embryos, morulae and blastocysts, and long-term lack of iron or copper increased apoptotic blastomeres. There might has a transition of primary demand for trace element iron or copper utilized by zygotes from cytoplast to culture medium after 8-cell of in vitro zygotes culture.
5. Determined lower osmotic pressure (0.075 M KCl) duration optimal for breaking granulosa cell membrane, and determined its effects on reconstructed embryos formation, reconstructed embryos developmental potential and cells apoptosis of blastocysts in bovine nuclear transfer by intracytoplasmic injection. Enucleated bovine oocytes were divided into five groups according to duration of 0.075 M KCl to granulosa cells: Os (control), 30s, 60s, 90s and 180s. Typical apoptotic nuclei was shown by staining of granulosa cells with DNA-binding fluorochrome Hoechst 33342. Different duration of 0.075 M KCl significantly increased the formation of reconstructed embryos than control. The treatment of 180s had significantly lower rate of blastocysts than other groups, and nuclei of blastocysts from 180s duration of 0.075 M KCl showed typical apoptosis morphology. These results suggested that lower osmotic pressure to bovine granulosa cells accelerated the formation of reconstructed embryos in nuclear transfer of intracytoplasmic injection, and long duration of lower osmotic pressure resulted in low blastocysts rate and high cells apoptosis rate of blastocysts.
引文
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