鱼类体细胞移核胚胎发育影响因素的探讨

将脱膜后的泥鳅 (Misgurnusanguillicaudatus)受精卵置于不同孵化液中孵化 ,结果表明 ,孵化液 1×Holtfreter中正常鱼苗和总鱼苗的孵化率最高 ,分别为 5 4 8%和 5 9 2 %。 1/ 2×Holtfreter、 1/ 10×Holtfreter、曝气冷开水和改变孵化液处理 ,正常鱼苗的孵化率差别不大 ,分别为 48 0 %、 49 6 %、 5 0 0 %和 48 8%。曝气冷开水畸形率最高 ,为 8 8%。PBS不适合用作孵化液。机械损伤对胚胎的孵化率无明显影响 (73 39%vs 75 70 % ) ,但对胚胎发育有明显的致畸作用 (8 0 6 %vs 0 )。以雌核发育鳙 (Aristichthysnobilis)尾鳍培养细胞作供体 ,泥鳅未受精卵作受体 ,微卫星标记分析表明 ,移核胚胎的核来源于供体核 ,受体卵中未除去的核被排斥。供体细胞培养代数严重影响移核胚胎的发育率。随着培养代数的增加 ,移核胚胎的发育率逐渐降低。继代核移植可提高移核胚胎的发育率 ,尤其是第 1次体细胞继代核移植 ,晚期囊胚的发育率急剧上升 (4 3 84%vs 7 6 9% ) ,说明继代核移植可促进受体卵对供体核的再程序化。泥鳅和大鳞副泥鳅 (Paramisgurnusdabryanus)未受精卵作受体 ,其移核胚胎发育率无明显区别 (7 6 9%vs 7 0 2 % )。     

人工诱导大鳞副泥鳅雄核发育二倍体克隆鱼的产生

首次获得人工诱导雄核发育二倍体大鳞副泥鳅克隆鱼。方法是:用紫外线210mJ/cm~2辐射浸泡在人工合成卵巢液中的泥鳅卵,使其染色体遗传失活,再与大鳞副泥鳅精子“受精”。在室温26℃条件下“受精”后15min开始,每隔2min一组,将“受精卵”置于39℃温水中热休克处理2min,以阻止第一次有丝分裂的发生,结果表明:二倍诱导率距“受精”后15～19min和27～29min出现两个高峰,最高二倍诱导率为61.1％,经染色体和形态特征鉴定表明,鱼苗为大鳞副泥鳅雄核发育二倍体克隆鱼,其染色体数(2n=48)和外部形态均与父本相同。     

人工诱导泥鳅与大鳞副泥鳅雄核发育单倍体子代的RAPD分析

用３０个经过筛选的随机引物对３组泥鳅雄核发育单倍体、２组大鳞副尼鳅雄核发育单倍体及其相应亲本进行了ＲＡＰＤ分析。结果表明,雄核发育倍体子代与其父本的ＲＡＰＤ谱带相似率为９７．０％－９７．８％,与母本相似率为３０．３％－５９．５％,子代中的非亲本谱眩为０－０．０２９,极少母本特异谱带。这一结果说明雄性发育鱼类单倍体的遗传信息主要来自父本,且存在着个体差异,雄核发育子代存在ＤＮＡ变异和母本ＤＮＡ非特异带,但并非其必要条件。     

泥鳅和大鳞副泥鳅卵雌核的紫外辐射灭活条件筛选

采用紫外线对泥鳅和大鳞副泥鳅卵雌性原核进行干法灭活以及在合成卵巢液、TC-199溶液、Ringer氏溶液和Holtfreter氏溶液中灭活,结果表明:当辐射剂量为90-180mJ/cm~2时,卵子在四种溶液中的单倍体诱导率均较高。合成卵巢液组的最佳辐射剂量为120-180mJ/cm~2;TC-199和Ringer氏溶液组的最佳辐射剂量为120mJ/cm~2;原液使用Holtfreter氏溶液效果欠佳,干法灭活效果最差。在上述各辐射剂量下,畸形苗经染色体组鉴定92％为雄核发育单倍体。综合评价,灭活效果依次是:人工合成卵巢液>TC-199溶液>Ringer氏溶液>Holtfreter氏溶液>干法。     

转基因红鲤体细胞的核移植

以F4代转hGH基因红鲤体细胞（肾脏和尾鳍）及培养18代的F4代转hGH基因红鲤尾鳍细胞为核供体,泥鳅或黄河鲤成熟卵为受体,进行了核移植,以探讨外源F4代转基因鱼体外源基因的分布与存在形式,稳定性和克隆转基因鱼的可能性。F4代红鲁肾脏细胞核与泥鳅卵配合的核移植胚胎有12．4％发育到囊胚,0．33％发育到神经胚;F4代尾鳍细胞核移入泥鳅卵后的重组胚发育到囊胚,神经胚、肌节期和肌肉效应期的胚胎分别为24．5％、0．3％、0．2％和0．1％;对照卵无发育。F4代红鲤尾鳍培养细胞与黄河鲤卵子配合的重组胚胎有50．53％发育到囊胚,5．69％发育到原肠胚,0．53％发育到神经胚,0．4％发育到肌节期。说明由于同种细胞核与卵细胞的相容性高于异种核卵的相容性,早期发育率高;而由于培养细胞的异倍化,后期的发育率降低。用PCR技术对供体鱼不同个体及同一体不同组织外源基因检测,结果100％个体为阳性鱼,而且不同组织的阳性率也是100％,说明外源基因均匀分布在不同组织中。无论F4代转基因鱼的肾脏细胞、尾鳍细胞还是培养的尾鳍细胞作核移植供体,核移植胚胎中hGH基因的检出率为100％。说明F4代转基因红鲤个体不同细胞都存在hGH基因,而且经长期培养不会丢失。表明F4代转基因红鲤中的外源hGH基因已基本稳定,体细胞核移植可以作为获得同质化转基因鱼的有效手段,但核移植效率还很低。另外还讨论了核质的相容性、细胞周期的协调、染色体的变异等因素对核移植的影响。     

人工诱导鱼类三倍体的试验

本试验利用大鳞副泥鳅的受精卵为材料,温度处理诱导大鳞副泥鳅同源三倍体,获得三倍体的胚胎和幼鱼。通过受精细胞学观察,初步提出了人工诱导鱼类三倍体过程中,染色体加倍的几种细胞学途径。     

人工诱导泥鳅雌核发育的细胞学研究和诱导参数优化

为培育泥鳅(Misgurnus anguillicaudatus)全雌品种,研究通过细胞荧光染色后进行细胞学观察确定人工诱导泥鳅雌核发育的热休克起始时间,并进一步利用核型和流式细胞分析等方法对人工诱导雌核发育泥鳅进行鉴定。结果显示,二倍体泥鳅受到灭活的鲤(Cyprinus carpio)精子刺激后,第二极体与卵核分开的时间在人工授精后3—5min;卵子在人工授精后3.5min后再热休克2min的诱导孵化率达到10.26%。通过分析野生型泥鳅胚胎、人工诱导雌核发育泥鳅胚胎、泥鳅×鲤杂交胚胎和单倍体泥鳅胚胎的发育,发现部分雌核发育的胚胎可以正常发育,而杂交胚胎和单倍体胚胎会出现明显的发育障碍。核型和流式细胞检测分析表明利用本研究获得的热休克参数进行处理可得到二倍体雌核发育子代,二倍率达64.71%。研究从细胞学层面获得泥鳅雌核发育的诱导参数,可为利用人工诱导雌核发育开展全雌泥鳅育种提供指导。     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

转人t-PA指形区缺失基因的山羊体细胞核移植及其继代核移植

为了探索转基因体细胞核经连续核移植后的发育潜力,以转人组织型纤溶酶原激活剂(t-PA)指形区缺失基因的山羊胎儿成纤维细胞为核供体,MII期的卵母细胞质为核受体,利用胞质内注射法构建原代核移胚胎(G0),并进行了原代核移植胚胎的继代核移植研究。比较原代和继代核移植胚胎在体外发育能力上的差异;在G1、G2代核移植试验过程中,比较了供体胚胎细胞的发育阶段对核移植胚胎体外发育的影响。结果表明,原代核移植胚胎的卵裂率(76.45%±1.17%)与继代核移植胚胎的卵裂率(72.18%±1.97%,76.05%±2.38%,75.99%±2.84%)无显著性差异(P>0.05)。但原代核移植胚胎的桑葚胚率(47.20%±2.93%)、囊胚率(11.00%±1.42%)显著高于G1、G2、G3代核核移植胚胎的桑葚胚率(34.99%±2.66%,28.23%±2.00%,23.34%±1.99%)、囊胚率(3.87%±0.67%,2.08%±1.66%,0);在G1、G2中,当用16-细胞期核移植胚胎作为核供体时的桑葚胚率(29.57%±1.53%,24.43%±1.87%)、囊胚率(1.96%±1.31%,2.01%±1.34%)低于用32~64-细胞时期的核移植胚胎的桑葚胚率(34.32%±1.31%,29.76%±1.66%)、囊胚率(3.86%±1.03%,3.48%±0.34%),但无显著性差异(P>0.05)。由此得出结论:转基因体细胞核移植胚胎不宜进行多代克隆;胞质内注射法构建核移植胚胎,用32~64-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率高于用16-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率。     

两种泥鳅中PdSox8和PdSox9基因的染色体定位

应用染色体原位杂交技术,以地高辛标记的大鳞副泥鳅ＰｄＳｏｘ８和ＰｄＳｏｘ９基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅染色体组中的定位。结果表明,在大鳞副泥鳅中ＰｄＳｏｘ８、ＰｄＳｏｘ９分别于端部着丝粒染色体第４和第２号即１４和１２染色体上,信号位点距着丝粒的相对距离分别为４０．２％和６７．５％,在泥鳅中,则分别位于ｔ９、ｔ６上,信息位点距着丝粒的相对蹁分别为５８．３％和３０．８％。     

Transplantation of blastula nuclei to non-enucleated eggs in the medaka, Oryzias latipes

Studies of nuclear transplantation were conducted to establish methods for the production of clones of fish, using a small laboratory fish, medaka, Oryzias latipes. As the first step of the study, single-blastula nuclei of an inbred strain with the wild-type body color were transplanted into non-enucleated unfertilized eggs of an outbred orange red strain. Of 845 operated eggs, 45 hatched into fry exhibiting the wild-type body color, one of the donor markers. Twenty-seven of these nuclear transplants grew to the adult stage and clearly exhibited external secondary sexual characteristics. Fourteen were females and 13 were males. The allozyme analysis of phosphoglucomutase, measurements of relative DNA content by microfluorometry and chromosome counts consistently indicated that the nuclear transplants were triploids that originated from both the diploid donor nuclei and the haploid recipient pronuclei. In the crossing experiments between the nuclear transplants and the orange-red strain, most of the male nuclear transplants were sterile, whereas one male produced a viable offspring with wild-type body color. All of the female nuclear transplants were sterile. Macroscopic observations of their gonads showed that the testes appeared normal and the ovaries appeared degenerated. These features of the reproductive potential and the morphology of gonads also indicated that the nuclear transplants were triploids. These results demonstrated that a basic technique for nuclear transplantation in medaka was established.     

Nuclear function during early development of remote fish hybrids

The participation of paternal genome was studied in the development of remote hybrids obtained as a result of artificial insemination of the loach (Misgurnus fossilis) eggs by the sperm of aquarial Cyprinids (Brachydanio rerio, Danio malabaricus, Barbus tetrazona, Razbora heteromorpha, Carassius auratus) and Cobitids (Acanthophtalmus kuhlii). The hybrids obtained differed at the stage of hatching both from each other and from the loach by some morphological features. To study the function of heterologous nuclei, haploid nucleocytoplasmic hybrids were obtained by means of chromosome inactivation in the loach eggs by heavy doses of X-rays. The participation of paternal genome in development was estimated by comparison of the curves of viability of diploid and haploid hybrids with those of diploid, haploid and "anuclear" loach embryos. Patterns of mortality of embryos and larvae in each hybrid combination (percentage, stage) suggest the functioning of paternal genome already at the early stages of development. The activity of hybrid and heterologous nuclei was also estimated by the onset and the intensity of morphogenetic function which was determined by the time of embryonic death following nuclear inactivation at different stages. The onset of nuclear function in all hybrids coinsides with that in the loach, except B. rerio in which it occurs somewhat earlier. The data obtained prove the participation of paternal genes in development and maintenance of viability of embryos at all developmental stages beginning from the early ones (blastula).     

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.     

Developmental incompatibility between cell nucleus and cytoplasm as revealed by nuclear transplantation experiments in teleost of different families and orders

Teleosts from different families and orders were used as materials for nuclear transplantation experiments. (1) The nuclei of goldfish (Carassius auratus, family Cyprinidae, order Cypriniformes) were transplanted into the enucleated egg cytoplasm of loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes) and vice-versa. (2) The nuclei of Tilapia (oreochromis nilotica, order Perciformes) were transplanted into the enucleated egg cytoplasm of goldfish (Carassius auratus, order Cypriniformes). The chromosome number of the nucleus donor fish is different from that of the cytoplasmic recipient fish in each of the two combinations. In the first case, only a few early nucleo-cytoplasmic hybrid (NCH) larval fish were obtained in each combination. In second case, even though a high percentage of NCH blastulas were also obtained, the majority of them died at the same developmental stage, except a few which survived until early gastrula stage. The examination of the metaphase chromosome figures of the NCH blastulas or embryos obtained in all three combinations indicated that they were of nucleus-donor type. The developmental rates of all the NCH eggs were similar to those of cytoplasmic-recipient type. Scanning electronmicroscopy examination showed that the morphology of NCH blastula cells, which were obtained from the combination of Tilapia nucleus and goldfish cytoplasm, manifested obviously abnormal features and the cells were arrested at different stages of cell disintegration. Two-dimension polyacrylamide gel electrophoretograms of the homogenates of Tilapia, goldfish and their NCH blastula cells showed that the protein synthetic pattern of NCH blastula was similar to that of Tilapia nucleus type. The results of experiments which failed to obtain NCH adult fish in all three combinations can be explained as a result of developmental incompatibility between the donor nucleus and the enucleated recipient egg cytoplasm, which were from distantly related fish species. And the chromosome numbers of all the component fish of the three combinations which were examined in the experiment and shown to be quite different from each other in the tested fish, should not be overlooked as one of the essential factors causing the developmental incompatibility in NCH fish in this experiment.     

Production of androgenetic diploid rainbow trout

Haploid androgenesis was induced in rainbow trout (Salmo gairdneri) when eggs were irradiated with 60Co gamma radiation prior to fertilization. Diploidy was restored to the androgenetic haploid zygotes by suppression of first cleavage division using hydrostatic pressure. Peak survival in the androgenetic diploid lots (32.5-38.9 percent of control) occurred when a pressure shock of 9000 pounds per square inch lasting from one to three minutes was applied to the eggs 345 minutes post-fertilization. Chromosomal analysis confirmed diploidy in the androgenetic individuals and suggested that YY rainbow trout are viable to at least the "eyed stage" of development. Inheritance patterns at two loci confirmed all-paternal inheritance. The relatively high yields of completely homozygous androgenetic rainbow trout and the potential for the use of androgenesis in the production of inbred lines and in genetic studies indicate that androgenesis may become a valuable tool in fish research and breeding.     

Expression of GFP in nuclear transplants generated by transplantation of embryonic cell nuclei from GFP-transgenic fish into nonenucleated eggs of medaka, Oryzias latipes

In order to investigate whether foreign genes can be used as genetic markers of donor nuclei in fish nuclear transplantation, expression of the GFP gene derived from donor nuclei was examined in nuclear transplants in medaka (Oryzias latipes). Embryonic nuclei were obtained from blastula embryos produced by crossing of transgenic fish of the wild-type strain heterozygous for the GFP gene with nontransgenic ones or by mutual crossing between transgenic fish. The GFP gene was driven by the promoter of the medaka elongation factor gene, EF-1alpha-A, which is known to induce GFP expression in many tissues except for the muscle in the transgenic fish. The nuclei were transplanted into nonenucleated unfertilized eggs of the orange-red strain. Adult nuclear transplants were successfully obtained at the rate of about 2% of the operated eggs. They were triploid and had no reproductive potential. The GFP gene was expressed in embryos, fry, and adults of nuclear transplants in a pattern similar to that in the transgenic fish. These results indicate that GFP is useful as a foreign genetic marker of donor nuclei in fish nuclear transplantation.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

Morphometric characteristics and reproductive capacity of nuclear transplants derived from embryonic cells of loach, Misgurnus anguillicaudatus

Nuclear transplants of loach were produced by transplantating blastula cell nuclei into nonenucleated unfertilized eggs, using recipient eggs and donor cells distinguished by different polymorphic microsatellites. Of the total of 2,847 operated eggs, 143 hatched and 119 developed to the feeding larval stage. For 15 nuclear transplants (i.e., 11.1-year-old fish and 4.2-year-old fish) that survived up to the adulthood, DNA analysis and karyotype analysis were performed. Results showed that, of the 15 fish, 14 had only a nucleus derived from the donor; moreover, 12 were diploids, 1 was a triploid, 1 was a tetraploid, and 1 was a diploid-tetraploid mosaic with both donor and recipient nuclei. For the 12 fish with only a 2n donor nucleus, morphometric analysis was performed, and two female fish and two male fish were mated with normal fish. The fish with only a 2n donor nucleus were determined to be morphometrically identical to normal fish: they had normal gametogenesis and were able to reproduce. Currently, nuclear transplantation technology is beginning to be adopted in fisheries. Biological information on nuclear transplants obtained in this study can be used in the development of nuclear transplantation technology.     

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.