Dynamics of lamin A/C in porcine embryos produced by nuclear transfer

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning, which is based on human somatic cell nuclear transfer, is one of our major research objectives. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage, and preparation method of donor cells on embryo development remain unclear. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast, and cumulus cells. The cumulus cell embryos showed significantly higher development rates than the other two (P < 0.05). The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference. Also, fluorescence in situ hybridization (FISH) was conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude that (1) cloned embryos can be constructed through human-rabbit interspecies nuclear transfer; (2) different kinds of somatic cells result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; (3) refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH. Translated from Zoological Research, 2005, 26(4): 416–421 [译自: 动物学研究]     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.     

Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum-starved skin cells were electrofused to enucleated oocytes, activated 1-5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1-2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6-56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus-like structure (PN), whereas, 64.5-78.3% of embryos activated 1-2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3-21.7%) compared to those of others (0-8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos.     

Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle

To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2–6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA‐types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast‐cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes. Mol. Reprod. Dev. 54:24–31, 1999. © 1999 Wiley‐Liss, Inc.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.     

Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.     

Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation

We compared developmental potential of somatic cell nuclear transfer (NT) embryos and postnatal survivability of cloned calves produced by two different fusion and activation protocols. As donor cells for NT, bovine cumulus cell-derived cultured cells of passage 5 were used following culture in serum-starved medium for 5-7 days. Enucleated oocytes were fused with donor cells at 21 or 24 hr post maturation. NT embryos fused at 21 hr were activated chemically 3 hr after fusion (DA group) and embryos fused at 24 hr were activated chemically immediately after fusion (FA group). Chemical activation was accomplished by calcium ionophore for 5 min and cytochalasin D + cycloheximide for 1 hr then cycloheximide alone for 4 hr. After in vitro culture in IVD101 medium for 7 days, embryo transfer was performed. Fusion rates were 86 and 84% in the DA and FA groups, respectively. Developmental rate to the blastocyst stage of NT embryos in the DA group was higher than in the FA group (42% vs. 28%). Pregnancy rate did not differ significantly between the DA and FA groups (11/13 and 5/7 at day 35), and 13 cloned calves (including 1 set of twins from a single embryo transfer) were born. High rates of postnatal mortality were observed in both groups. These results suggest that the DA method improves in vitro developmental potential of NT embryos, but the timing of fusion and chemical activation does not affect the pregnancy rate and the survivability of cloned calves.     

不同供体细胞及其处理对猪核移植重构胚体外发育的影响

系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40~44 h的猪卵母细胞去核后, 将经血清饥饿(0.5%FBS)培养2~9天、0.1 mg/L Aphidicolin(APD)培养+0.5% FBS培养2~9天或一般培养法(10% FBS)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞, 直接注射到去核的卵母细胞质中, 或注射到卵周隙中, 再经电融合(100 V/mm, 30 [mu]s, 电脉冲1次)构建重构胚。重构胚以钙离子载体A23817 或电脉冲结合6-DMAP 激活处理, 体外培养6天。耳皮成纤维细胞和颗粒细胞经0.1 mg/L APD + 0.5% FBS培养处理后的重组胚卵裂率, 均高于血清饥饿和一般培养处理的同种供体细胞(P<0.01)。卵丘细胞、颗粒细胞经0.1 mg/L APD + 0.5% FBS处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P<0.05)。以猪颗粒细胞为核供体时, 电融合法的重构胚分裂率显著高于胞质内注入法(P<0.05), 但囊胚发育率无显著差异(P>0.05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞, 其具有正常二倍染色体的细胞比例均无显著差异(P>0.05); 以这2种细胞不同培养代数做供体进行核移植时, 各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P>0.05)。这些结果表明: (1) 猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定; (2) 0.1 mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果, 血清饥饿培养则无明显效果; (3) 猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞, 核移植后都能得到体细胞克隆的囊胚, 但前者的效果略优于后者, 且其核移植效果不受供核细胞培养代数的影响; (4) 电融合核移植胚胎的发育率高于胞质内直接注入法, 但两者的总体效率相近。     

Chromatin in early mammalian embryos: achieving the pluripotent state

Abstract Gametes of both sexes (sperm and oocyte) are highly specialized and differentiated but within a very short time period post-fertilization the embryonic genome, produced by the combination of the two highly specialized parental genomes, is completely converted into a totipotent state. As a result, the one-cell-stage embryo can give rise to all cell types of all three embryonic layers, including the gametes. Thus, it is evident that extensive and efficient reprogramming steps occur soon after fertilization and also probably during early embryogenesis to reverse completely the differentiated state of the gamete and to achieve toti- or later on pluripotency of embryonic cells. However, after the embryo reaches the blastocyst stage, the first two distinct cell lineages can be clearly distinguished—the trophectoderm and the inner cells mass. The de-differentiation of gametes after fertilization, as well as the differentiation that is associated with the formation of blastocysts, are accompanied by changes in the state and properties of chromatin in individual embryonic nuclei at both the whole genome level as well as at the level of individual genes. In this contribution, we focus mainly on those events that take place soon after fertilization and during early embryogenesis in mammals. We will discuss the changes in DNA methylation and covalent histone modifications that were shown to be highly dynamic during this period; moreover, it has also been documented that abnormalities in these processes have a devastating impact on the developmental ability of embryos. Special attention will be paid to somatic cell nuclear transfer as it has been shown that the aberrant and inefficient reprogramming may be responsible for compromised development of cloned embryos.     

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.     

Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer

Fragmentation occurs during early developmental stages of electrically activated oocytes and nuclear transfer (NT) embryos. It might contribute to the low developmental rate of porcine NT embryos. The present study was conducted to investigate whether the addition of sugars such as sorbitol or sucrose suppresses fragmentation and supports the development of electrically activated oocytes and NT embryos. The activated oocytes were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with sorbitol or sucrose for 2 days after electric activation, and then cultured in the PZM-3 for the remaining 4 days. The osmolarities of PZM-3, PZM-3 supplemented with 0.05 or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose were 269 +/- 6.31, 316 +/- 3.13, 362 +/- 4.37, and 315 +/- 5.03 mOsm, respectively. When parthenogentically activated oocytes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate were observed. Interestingly, addition of sugar to PZM-3 for 2 days reduced the fragmentation rate compared to PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 increased the fusion rate (84.2% +/- 6.07 vs. 95.1% +/- 2.52), cleavage rate (67.6% +/- 5.80 vs. 77.3% +/- 3.03), and developmental rate to the blastocyst stage (10.2% +/- 0.79 vs. 19.4% +/- 1.77). There was no significant difference between treatments for the number of the blastocysts. In addition the fragmentation rate was reduced compared to PZM-3 without sorbitol (26.1 +/- 4.30 vs. 14.5 +/- 1.74). In conclusion, increasing the osmolarity of PZM-3 through addition of either sorbitol or sucrose for 48 hr increased the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate through increasing osmolarity.     

Electrofusion parameters for nuclear transfer predicted using isofusion contours produced with bovine embryonic cells

Electrofusion is a valuable technique for the nuclear transfer procedure. An enucleated oocyte is electrofused with a blastomere to create a nuclear transfer embryo. The present study constructed isofusion contours after the electrofusion of identical coupled cells that characterized all the bovine embryonic cell types used in nuclear transfer. The intersection of isofusion contours for enucleated oocytes and blastomeres provided the parameters for electrofusion during nuclear transfer. Blastomeres isolated from in vitro produced embryos 3–6 days after (in vitro fertilization) were electrofused with oocytes enucleated by centrifugation (85, 87, 89, and 73% electrofusion, respectively). The cleavage (46, 40, 37, and 28%, respectively) of the nuclear transfer embryos produced a trend that decreased as the age of the blastomeres increased. The isofusion contours provided information about the interaction between different cell types in an electric field, and gave precise electrofusion parameters for a range of bovine embryonic cell types used in nuclear transfer. © 1996 Wiley-Liss, Inc.