小鼠体细胞核移植和胚胎分割相关问题研究
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摘要
细胞核移植和胚胎分割是目前生产哺乳类克隆动物最重要的两种方法。细胞核移植是以早期胚胎细胞、胚胎干细胞或体细胞核作核供体,移入去核的卵母细胞中,使供体核在受体胞质中去分化,启动重构胚发育程序的过程。体细胞核移植研究不仅是发育生物学中一个极为重要的理论问题,而且是动物繁殖、人类治疗性克隆中的重要应用课题。该项研究目前尚处于初始阶段,其中许多理论问题尚不明确,技术程序很不成熟,是目前各国学者竞相突破的焦点问题。胚胎分割即分割2-细胞期至囊胚期不同阶段胚胎,产生同卵孪生或多生后代的克隆方法。对于小鼠来说,植入前胚胎分割成功率及胚胎分割后的发育率均较低,其影响因素尚需研究和探索。为此我们对动物克隆中体细胞核移植和胚胎分割中几个关键环节及其影响因素进行了较为系统和深入的研究。
一、动情周期时相对小鼠超排卵母细胞质量和孤雌激活后体外发育的影响 体细胞核移植中,卵母细胞作为胞质受体,其数量和质量直接影响体细胞核移植的整体效果。以往研究证实,地鼠、大鼠及其他家畜动物的诱导超排卵实验中,动情周期对超排效果均具有重要影响,但在小鼠的超排诱导实验中,人们却一直忽略了动情周期的影响。我们用孕马血清促性腺激素作为外源生殖激素对处于动情前期、动情期、动情后期及间情期小鼠进行超排卵诱导,收集并计算形态正常卵母细胞比率,并将正常卵母细胞用SrCl_2孤雌激活后体外培养,探讨动情周期时相对小鼠超排卵母细胞质量和孤雌激活后体外发育的影响。结果显示,动情前期和动情期注射组获得的形态正常卵母
Nuclear transfer and embryo splitting are two most important methods to produce mammalian clones. Nuclear transfer is a process,during which the nuclus derived from early embryo blastomeres,embryonic stem cells or somatic cells is transferred to enucleated oocyte and reprogrammed by ooplasma to support the development of reconstructed embryos. It is of particular impotance to study the problems related to nuclear transfer not only in the exploring of fundamental theory in the developmental biology but also in the application of animal reproductive clone and human therapeutic clone and so on . Nuclear transfer has been become one of the most attentioned questions in scientific fields. The initial successes have been made in this fields in the last decades,but the elements involved in procedure of nuclear transfer and the underlying fundamental theories are to be clarified.Embryo splitting is a method to produce monozygotic twins from preimplantation embryos by mechanical splitting. The influence factor involved in embryo splitting procedures should be to elucidate. And the efficiency of preimplantation embryo splitting should be improved. So we make a through on study the elements involved in the key steps in the nuclear transfer and embryo splitting. The influence of the phases of female estrus cycle on superovulation in mice The quanlity and quannity of oocyte as recipient cytoplasm has a direct influence on the whole efficiency of nuclear transfer.Domestic animals,hamsters and rats are induced to
superovulation at a particular phase of the estrus cycle to maximize the number of oocytes/embryos collected.However, superovulation in mice has been induced always on random days of the estrus cycle. To investigate the effect of the phase of mouse estrus cycle on quality of oocytes collected after exogenous ovarian stimulation.Superovulation was induced by a priming injection of PMSG at different phase(proestrus, estrus, metestrus or diestrus) of the estrus cycle followed after 48-hr interval by HCG injection. The rate of normal oocytes was assessed. The oocytes retrieved from female mice at different phase of estrus cycle were pathenogeneticly activated by SrCl_2,and cultured in vitro to investigate the effect of the phase of oestrus cycle on in vitro development potential of morphological normal oocytes following parthenogenetic activation.The results show that after administration of PMSG, the mice at the phase of estrus or proestrus gave the best quality of ovulated oocytes, and the highest percentage of blastocyst developing after artificial activation of oocytes.On the country.the mice at the phase of diestrus provide the worst result.These results indicated that administration of exogenous gonadotropins in mice should be synchronized with the innate estrus cycle of female to optimize the quality of the oocytes harvested from oviduct and their development competence after pathenogenetic activation.? Study on in vitro cultruring of mouse preimplantation embryos In vitro culturing of mammalian embryos is not only a key step of the embryonic engineering,but also a important method to investigate the mechanisms of embryonic development .To establish a stable and appropriate culture system is very important for improving the developmental quality of embryos in vitro and for raising the efficiency of the embryonic engineering.To improve the chemical composition of the medium used in embryo culture according to the in vivo microenvironment has became a important respect of embryonic research.In the present study,we established a chemical defined medium applied to the study of defined medium applied to the study of developmental regulating factors for early embryos and compared the effect of this medium with that of the traditional medium on in vitro development of mouse early embryos,and we evaluated the effect of CZB medium supplemented respectively with EGF and insulin on in vitro development of
mouse early embryos.The results show that l,the CZB medium supplemented with 0.05mg/ml PVA(polyvinyl alcohol),0.5 X EAA(essential amino acid) and 0.5 X NEAA(non-essential amino acid),the chemical defined medium,can not only prevent the early embryos from adhesion to each other effectively,but also support the preimplantation embryos development in vitro to blastocysts.lt is proved that this medium is appropriate for the study of developmental mechanism of early mouse embryos.2,Supplementation of both CZB medium with EGF at the concentration of O.lng/ml can improve the developmental potential of mouse preimplantation embryos in vitro by means of stimulating the cell differentiation,but the concentration of lOOng/ml of EGF is cytotoxic and can damage the early embryos.3,Supplementation of CZB medium with insulin at the concentration of 0.05ng/ml and glucose at the concentration of 5.56mmol/L caused a significant increase in the rate of expanded and hatched blastocysts and the cell number of blastocysts in both normally originated and IVF-originated embryos.Supplementation of the chemical defined medium with insulin and glucose has the same effects mentioned above to both normally and IVF-originated embryos,but the concentration of insulin supplemented is much less than that in CZBmedium containing BSA.? Study on embryo splitting In the present study,the developmental potential,in vitro and in vivo of splitted embryos,the possible influence elements and their mechanism on it were explored to increase the rate of survival and development of splitted embryos.The major works are as follows. 1,The single blastomeres from 2-8 cell embryos(l/2,1/4,1/8), half-embryos from 2-8cell embryos(l/2,2/4,4/8)and different number of blastomeres from 8-cell embryos(l/8,2/8,4/8,6/8)were obtained by mechanical methods and cultured in CZB medium after inserted into empty zona pellucida.When the splitted embryos developed to blastocyst stage,the percentage of blastocyst and the cell number in each blastocyst were examed to explore the developmental potential of blastomeres derived from precompacted mouse embryos.The results show that the mice early the embryo is the higher the developmental potential of its single blastomere is (l/2>l/4>l/8),the earlier the embryos is ,the higher the developmental potential of its
half-embryos is (4/8>2/4>l/2),and the more blastomeres the splitted embryo contains,thehigher the developmental potential of the embryo is (6/8>4/8>2/8>l/8).2,The isolatedsingle blastomeres (l/2,l/4,l/8)and half-embryos(l/2,2/4,4/8)were cultured in differentconditions,to investigate the effects of the embryo density.of zona pellucida and of thesupplementation of insulin on the developmental potential of the splitted embryos.Theresult shows that appropriately increasing the embryo density(from 5/50 u 1 to 25/50 ul)and supplementation of insulin (0.05 u g/ml)and glucose(5.56mmol/L)can significantlyelevate the developmental potential in vitro of the splitted embryos from 2-8cellembryos,but the zona pellucida (with or without) has no significant effect on thedevelopment of the single blastomere derived from 2-cell embryos.3,Mouse morula andblastocyst were splitted with micromanipulater.The half embryos were cultured in vitro toblastocyst stage and some of them were transferred into the uteri of the pseudopregnantedmouse on day 3.5.The preimplantation development potential in vitro and in vivo of thehalf embryos were examed.The result shows that significantly more half embryos derivedfrom the blastocysts developed in vitro to the blastocyst stage than those derived from themorula,and after transferred into the recipient uteri,the pregnancy rates of the halfembryos from both morula and blastocysts, however,have no significantdifference.4,Leptin is a regulating protein involving in the reproductive course includingpreimplantation development, implantation and pregnancy. We examed the distributionfeatures of leptin in both normal embryo and splitted embryos and the difference ofexpression between the normal blastocysts and the half embryo derived blastocysts withthe CLSM(confocal laser scanning microscopy)to study the relation between theexpression of the leptin in half embryos and their development potential after transfer intorecipients.The result shows that the leptin has a same distribution pattern in normal andsplitted embryos.In the preblastocystic stage,the expression intensity of leptin in differentblastomeres is unequal,but in the blastocysts the leptin is mainly distributed in thetrophoblast.The expression intensity of leptin in the blastocysts derived from halfembryos is significantly lower than that in normal blastocysts.This may be one of thecause of lower developing potential of the half embryos after transferred into recipients.
一、动情周期时相对小鼠超排卵母细胞质量和孤雌激活后体外发育的影响 体细胞核移植中,卵母细胞作为胞质受体,其数量和质量直接影响体细胞核移植的整体效果。以往研究证实,地鼠、大鼠及其他家畜动物的诱导超排卵实验中,动情周期对超排效果均具有重要影响,但在小鼠的超排诱导实验中,人们却一直忽略了动情周期的影响。我们用孕马血清促性腺激素作为外源生殖激素对处于动情前期、动情期、动情后期及间情期小鼠进行超排卵诱导,收集并计算形态正常卵母细胞比率,并将正常卵母细胞用SrCl_2孤雌激活后体外培养,探讨动情周期时相对小鼠超排卵母细胞质量和孤雌激活后体外发育的影响。结果显示,动情前期和动情期注射组获得的形态正常卵母
Nuclear transfer and embryo splitting are two most important methods to produce mammalian clones. Nuclear transfer is a process,during which the nuclus derived from early embryo blastomeres,embryonic stem cells or somatic cells is transferred to enucleated oocyte and reprogrammed by ooplasma to support the development of reconstructed embryos. It is of particular impotance to study the problems related to nuclear transfer not only in the exploring of fundamental theory in the developmental biology but also in the application of animal reproductive clone and human therapeutic clone and so on . Nuclear transfer has been become one of the most attentioned questions in scientific fields. The initial successes have been made in this fields in the last decades,but the elements involved in procedure of nuclear transfer and the underlying fundamental theories are to be clarified.Embryo splitting is a method to produce monozygotic twins from preimplantation embryos by mechanical splitting. The influence factor involved in embryo splitting procedures should be to elucidate. And the efficiency of preimplantation embryo splitting should be improved. So we make a through on study the elements involved in the key steps in the nuclear transfer and embryo splitting. The influence of the phases of female estrus cycle on superovulation in mice The quanlity and quannity of oocyte as recipient cytoplasm has a direct influence on the whole efficiency of nuclear transfer.Domestic animals,hamsters and rats are induced to
superovulation at a particular phase of the estrus cycle to maximize the number of oocytes/embryos collected.However, superovulation in mice has been induced always on random days of the estrus cycle. To investigate the effect of the phase of mouse estrus cycle on quality of oocytes collected after exogenous ovarian stimulation.Superovulation was induced by a priming injection of PMSG at different phase(proestrus, estrus, metestrus or diestrus) of the estrus cycle followed after 48-hr interval by HCG injection. The rate of normal oocytes was assessed. The oocytes retrieved from female mice at different phase of estrus cycle were pathenogeneticly activated by SrCl_2,and cultured in vitro to investigate the effect of the phase of oestrus cycle on in vitro development potential of morphological normal oocytes following parthenogenetic activation.The results show that after administration of PMSG, the mice at the phase of estrus or proestrus gave the best quality of ovulated oocytes, and the highest percentage of blastocyst developing after artificial activation of oocytes.On the country.the mice at the phase of diestrus provide the worst result.These results indicated that administration of exogenous gonadotropins in mice should be synchronized with the innate estrus cycle of female to optimize the quality of the oocytes harvested from oviduct and their development competence after pathenogenetic activation.? Study on in vitro cultruring of mouse preimplantation embryos In vitro culturing of mammalian embryos is not only a key step of the embryonic engineering,but also a important method to investigate the mechanisms of embryonic development .To establish a stable and appropriate culture system is very important for improving the developmental quality of embryos in vitro and for raising the efficiency of the embryonic engineering.To improve the chemical composition of the medium used in embryo culture according to the in vivo microenvironment has became a important respect of embryonic research.In the present study,we established a chemical defined medium applied to the study of defined medium applied to the study of developmental regulating factors for early embryos and compared the effect of this medium with that of the traditional medium on in vitro development of mouse early embryos,and we evaluated the effect of CZB medium supplemented respectively with EGF and insulin on in vitro development of
mouse early embryos.The results show that l,the CZB medium supplemented with 0.05mg/ml PVA(polyvinyl alcohol),0.5 X EAA(essential amino acid) and 0.5 X NEAA(non-essential amino acid),the chemical defined medium,can not only prevent the early embryos from adhesion to each other effectively,but also support the preimplantation embryos development in vitro to blastocysts.lt is proved that this medium is appropriate for the study of developmental mechanism of early mouse embryos.2,Supplementation of both CZB medium with EGF at the concentration of O.lng/ml can improve the developmental potential of mouse preimplantation embryos in vitro by means of stimulating the cell differentiation,but the concentration of lOOng/ml of EGF is cytotoxic and can damage the early embryos.3,Supplementation of CZB medium with insulin at the concentration of 0.05ng/ml and glucose at the concentration of 5.56mmol/L caused a significant increase in the rate of expanded and hatched blastocysts and the cell number of blastocysts in both normally originated and IVF-originated embryos.Supplementation of the chemical defined medium with insulin and glucose has the same effects mentioned above to both normally and IVF-originated embryos,but the concentration of insulin supplemented is much less than that in CZBmedium containing BSA.? Study on embryo splitting In the present study,the developmental potential,in vitro and in vivo of splitted embryos,the possible influence elements and their mechanism on it were explored to increase the rate of survival and development of splitted embryos.The major works are as follows. 1,The single blastomeres from 2-8 cell embryos(l/2,1/4,1/8), half-embryos from 2-8cell embryos(l/2,2/4,4/8)and different number of blastomeres from 8-cell embryos(l/8,2/8,4/8,6/8)were obtained by mechanical methods and cultured in CZB medium after inserted into empty zona pellucida.When the splitted embryos developed to blastocyst stage,the percentage of blastocyst and the cell number in each blastocyst were examed to explore the developmental potential of blastomeres derived from precompacted mouse embryos.The results show that the mice early the embryo is the higher the developmental potential of its single blastomere is (l/2>l/4>l/8),the earlier the embryos is ,the higher the developmental potential of its
half-embryos is (4/8>2/4>l/2),and the more blastomeres the splitted embryo contains,thehigher the developmental potential of the embryo is (6/8>4/8>2/8>l/8).2,The isolatedsingle blastomeres (l/2,l/4,l/8)and half-embryos(l/2,2/4,4/8)were cultured in differentconditions,to investigate the effects of the embryo density.of zona pellucida and of thesupplementation of insulin on the developmental potential of the splitted embryos.Theresult shows that appropriately increasing the embryo density(from 5/50 u 1 to 25/50 ul)and supplementation of insulin (0.05 u g/ml)and glucose(5.56mmol/L)can significantlyelevate the developmental potential in vitro of the splitted embryos from 2-8cellembryos,but the zona pellucida (with or without) has no significant effect on thedevelopment of the single blastomere derived from 2-cell embryos.3,Mouse morula andblastocyst were splitted with micromanipulater.The half embryos were cultured in vitro toblastocyst stage and some of them were transferred into the uteri of the pseudopregnantedmouse on day 3.5.The preimplantation development potential in vitro and in vivo of thehalf embryos were examed.The result shows that significantly more half embryos derivedfrom the blastocysts developed in vitro to the blastocyst stage than those derived from themorula,and after transferred into the recipient uteri,the pregnancy rates of the halfembryos from both morula and blastocysts, however,have no significantdifference.4,Leptin is a regulating protein involving in the reproductive course includingpreimplantation development, implantation and pregnancy. We examed the distributionfeatures of leptin in both normal embryo and splitted embryos and the difference ofexpression between the normal blastocysts and the half embryo derived blastocysts withthe CLSM(confocal laser scanning microscopy)to study the relation between theexpression of the leptin in half embryos and their development potential after transfer intorecipients.The result shows that the leptin has a same distribution pattern in normal andsplitted embryos.In the preblastocystic stage,the expression intensity of leptin in differentblastomeres is unequal,but in the blastocysts the leptin is mainly distributed in thetrophoblast.The expression intensity of leptin in the blastocysts derived from halfembryos is significantly lower than that in normal blastocysts.This may be one of thecause of lower developing potential of the half embryos after transferred into recipients.
引文
1. Lasserre A, Espinosa MB,Vitullo A.Superovulation in vesper mouse Calomys musculinus (Rodentia-Sigmodontinae). Biocell 1998, 22(3): 157
2. Chatot CL, Ziomek CA,Bavister BD,et al.An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro[J]. J Reprod Fert, 1989, 86: 679
3.施新猷.医学动物实验方法.北京:人民卫生出版社,1980,93,141
4. Edwards RG, Fowler RE. Superovulation treatment of adult mice: Their subsequent natural fertility and response to further treatment. J Endocrinol 1960, 21: 147
5. Edwards RG, Wilson ED, Fowler RE. Genetic and hormonal influences on ovulation and implantation in adult mice treated with gonadotrophins.J Endocrinal 1963, 26: 389
6. Fowler RE, Edwards RG. Induction of superovulation and pregnancy in mature mice by gonadotrophins. J Endocrinal 1957, 15: 374
7. Sakai N, Endo A. Effects of in-phase and out-phase ovulation induction on early mouse embryos. Reprod Toxicol 1988, 1: 189
8. Sakai N, Endo Potential teratogenicity of gonadotropin treatment for ovulation induction in the mouse offspring. Teratology 1987, 36: 229
9. Redina OE, Amstislavsky Sya, Maksimovsky LF. Induction of superovulation in DD mice zt different stages of the oestrous cycle. J Reprod Fertil 1994, 102: 263
10. Kanayama K, Shiiki Y. Scanning electron microscopic findings of cleaved unfertilized eggs ovulated by repeated administration of gonadotrophin. Res Commun Mol Pathol Pharmacol 2000, 107(3-4): 253
11. Kanayama K, Shiiki Y. Findings on nuclear staining using Hoechst dye in cleaved unfertilized eggs obtained by repeated superovulation with gonadotrophin. Res Commun Mol Pathol Pharmacol 2000, 107(1-2): 33
12. Loi P, Ledda S, Fulka J Jr, et al. Development of parthenogenetic and cloned
2. Chatot CL, Ziomek CA,Bavister BD,et al.An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro[J]. J Reprod Fert, 1989, 86: 679
3.施新猷.医学动物实验方法.北京:人民卫生出版社,1980,93,141
4. Edwards RG, Fowler RE. Superovulation treatment of adult mice: Their subsequent natural fertility and response to further treatment. J Endocrinol 1960, 21: 147
5. Edwards RG, Wilson ED, Fowler RE. Genetic and hormonal influences on ovulation and implantation in adult mice treated with gonadotrophins.J Endocrinal 1963, 26: 389
6. Fowler RE, Edwards RG. Induction of superovulation and pregnancy in mature mice by gonadotrophins. J Endocrinal 1957, 15: 374
7. Sakai N, Endo A. Effects of in-phase and out-phase ovulation induction on early mouse embryos. Reprod Toxicol 1988, 1: 189
8. Sakai N, Endo Potential teratogenicity of gonadotropin treatment for ovulation induction in the mouse offspring. Teratology 1987, 36: 229
9. Redina OE, Amstislavsky Sya, Maksimovsky LF. Induction of superovulation in DD mice zt different stages of the oestrous cycle. J Reprod Fertil 1994, 102: 263
10. Kanayama K, Shiiki Y. Scanning electron microscopic findings of cleaved unfertilized eggs ovulated by repeated administration of gonadotrophin. Res Commun Mol Pathol Pharmacol 2000, 107(3-4): 253
11. Kanayama K, Shiiki Y. Findings on nuclear staining using Hoechst dye in cleaved unfertilized eggs obtained by repeated superovulation with gonadotrophin. Res Commun Mol Pathol Pharmacol 2000, 107(1-2): 33
12. Loi P, Ledda S, Fulka J Jr, et al. Development of parthenogenetic and cloned