Recipient Ooplasm Research Articles

Rescue of oocytes recovered from postmortem mouse ovaries.

It is well known that the survivability of gametes of postmortem carcass was decreased as time passes after death. In this study, it was examined whether cytoplasmic replacement rescues the survivability of germinal vesicle stage (GV) oocytes of postmortem carcass in the mouse. Reactive oxygen species (ROS) levels and mitochondria numbers in GV oocytes of the dead mice stored at 4 degrees were significantly impaired after 44 h postmortem compared to the control (0 h). However, when kayoplasts of GV oocytes of postmortem carcass was transferred to recipient ooplasts (GV transfer), proportion of in vitro maturation (IVM), normal spindle morphology, in vitro and in vivo developmental ability after in vitro fertilization (IVF) of reconstituted oocytes was improved. Moreover, secondary follicle oocytes of postmortem carcass were developed, matured and fertilized in vitro and developed to go to term, when GV transfer was conducted at the GV phase. Thus, transfer of GV karyoplast recovered from postmortem carcass, which viability was decreased, into fresh GV recipient ooplasm, rescues survivability of reconstituted oocytes. It suggested the effective use of oocytes of dead animals in the mouse and this achievement must apply to other rare animal species, especially animals under control by human.

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

The perspective of the incompatible of nucleus and mitochondria in interspecies somatic cell nuclear transfer for endangered species.

Taking into account the latest Red List of the International Union for Conservation of Nature in which 25% of all mammals are threatened with extinction, somatic cell nuclear transfer (SCNT) could be a beneficial tool and holds a lot of potential for aiding the conservation of endangered, exotic or even extinct animal species if somatic cells of such animals are available. In the case of shortage and sparse amount of wild animal oocytes, interspecies somatic cell nuclear transfer (iSCNT), where the recipient ooplasm and donor nucleus are derived from different species, is the alternative SCNT technique. The successful application of iSCNT, resulting in the production of live offspring, was confirmed in several combination of closely related species. When nucleus donor cells and recipient oocytes have been used in many other combinations, very often with a very distant taxonomical relation iSCNT resulted only in the very early stages of cloned embryo development. Problems encountered during iSCNT related to mitochondrial DNA (mtDNA)/genomic DNA incompatibility, mtDNA heteroplasmy, embryonic genome activation of the donor nucleus by the recipient oocyte and availability of suitable foster mothers for iSCNT embryos. Implementing assisted reproductive technologies, including iSCNT, to conservation programmes also raises concerns that the production of genetically identical populations might cause problems with inbreeding. The article aims at presenting achievements, limitations and perspectives of iSCNT in maintaining animal biodiversity.

DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50% and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho +–Hd −) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho −–Hd +) or non-heated (Ho −–Hd −) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6–32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9–33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.

An Unknown Piece of Early Work of Nuclear Reprogramming in Fish Eggs

In a party held on February 19, 2010­ celebrating his scientific career of 60 years, Professor Hongxi Chen passed out to us a reprint of his early work of nuclear transplantation in fish eggs. The 80 year-old man told us that he had read a recent report about a group of scientists obtained 27 mice from iPS (induced pluripotent stem) cells derived from mouse skin fibroblasts 1. This result demonstrated that the nuclei of somatic cells, like that of undifferentiated embryonic cells, are pluripotent after genetic manipulation and reprogram in vitro. Indeed, successful animal cloning has been reported from many mammals, which demonstrates that the nuclei of donor cells, under influence of recipient ooplasm, are able to undergo reprogramming and restore their totipotency 2. It is now an accepted concept in the scientific field that the developmental potential of a differentiated nucleus and an undifferentiated nucleus is the same. But, this consensus does not come easily as professor Chen discussed. It actually took almost half a century of gradual improvement of the technology and accumulation of knowledge. Briggs and King (1952) 3 showed their ground-breaking work that normal tadpoles could be obtained through transplantation of nuclei of blastula cells to enucleated eggs in the frog Rana pipines. Since then many developmental biologists have studied developmental potentiality of the nuclei of amphibians using this technology. In 1966, Gurdon et al. 4 obtained several sexually mature individuals through transplanting dissociated intestinal epithelial nuclei from feeding tadpoles of Xenopus laevis into enucleated eggs of the Xenopus laevis. This study provides the first evidence that the differentiated somatic nuclei preserve their developmental totipotency. However, many investigators failed to obtain live animals beyond tadpoles stage from donor nuclei of various somatic cell. Thus, it remains controversial whether the differentiated somatic nuclei maintain their developmental totipotency. In 1982, a group of Chinese scientists, led by Hongxi Chen successfully obtained a sexually mature adult fish from a short-term cultured kidney cell nucleus of an adult crucian carp 5. This study provides further evidence that the differentiated somatic nuclei, even from the kidney of an adult, indeed preserve their developmental totipotency. However, due to the isolation in the early period of after the Cultural Revolution, this work was only reported in a Chinese journal and it has been largely unknown by scientists in the field. In 1997, Wilmut et al. 6 obtained the famous sheep “Dolly” from the nucleus isolated from a mammary gland cell, which proves again that the nucleus for fully differentiated cells are totipotent. Since then, the somatic cloning has achieved enormous successes in both basic and applied sciences, demonstrating a great and bright future. Looking back the history of the animal somatic cloning, we respect those who contributed great efforts in pioneering the fish somatic nuclear transplantation and demonstrated at first time that nuclei from adult kidney cells were able to undergo reprogramming and differentiation to form sexually mature adults. We believe it is necessary to translate their work, which was originally published in Chinese 5, into English and publish it in a hope more readers now have an opportunity to know this excellent, yet largely unknown work 7.

Preimplantation Development of Somatic Cell Cloned Embryos in the Common Marmoset (Callithrix jacchus)

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Production of Boer goat (Capra hircus) by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes

The present study reports a birth of goat clone produced by nuclear transfer of cultured and cryopreserved fibroblast cells into slaughterhouse-derived oocytes. The donors of somatic cells were fibroblasts derived from the ear skin of a Boer goat while the recipient ooplasm was &lt;I&gt;in vitro&lt;/I&gt; matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were cultured &lt;I&gt;in vitro&lt;/I&gt; and then the morphologically normal embryos were transferred to the surrogates. The reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural oestrus. Five of them were treated with hCG after transfer. Among them, one was pregnant and gave birth to a live kid. Due to the improper delivery aid, the cloned kid died accidentally after birth. The cloned kid was then anatomised to observe the viscera development, and the results showed that the organs were normal. Paraffin tissue slices were prepared and stained to ensure the viscera development further, and the results suggested that the organs also developed well in spite of incipient hydropericardium. The microsatellite analysis identified the cloning. It is suggested that the optimised nuclear transfer protocol and proper hCG treatment lead to the successful birth of a goat clone.

158 IN VITRO DEVELOPMENT OF WOOD BISON (BISON BISON ATHABASCAE) EMBRYOS BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P &lt; 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P &lt; 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

Tough beginnings: Alterations in the transcriptome of cloned embryos during the first two cell cycles

Cloned embryos produced by somatic cell nuclear transfer (SCNT) display a plethora of phenotypic characteristics that make them different from fertilized embryos, indicating defects in the process of nuclear reprogramming by the recipient ooplasm. To elucidate the extent and timing of nuclear reprogramming, we used microarrays to analyze the transcriptome of mouse SCNT embryos during the first two cell cycles. We identified a large number of genes mis-expressed in SCNT embryos. We found that genes involved in transcription and regulation of transcription are prominent among affected genes, and thus may be particularly difficult to reprogram, and these likely cause a ripple effect that alters the transcriptome of many other functions, including oxidative phosphorylation, transport across membrane, and mRNA transport and processing. Interestingly, we also uncovered widespread alterations in the maternal (i.e., non-transcribed) mRNA population of SCNT embryos. We conclude that gene expression in early SCNT embryos is grossly abnormal, and that this is at least in part the result of incomplete reprogramming of transcription factor genes.

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.

91 PRODUCTION OF A CLONED BOER GOAT (CAPRA HIRCUS) BY SOMATIC CELL NUCLEAR TRANSFER

We reported the birth of a goat clone produced by somatic cell nuclear transfer. The fusion and activation protocols of reconstructed oocytes and embryo transfer procedure were optimized. The donors of somatic cells were fibroblasts derived from ear skin of a Boer goat while the recipient ooplasm was in vitro-matured oocytes of Huanghuai white goat, an Anhui native goat species. The reconstructed embryos were activated by ionomycin, 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB) singly or simultaneously (termed as Ionomycin, Ionomycin+6-DMAP, and Ionomycin+6-DMAP+CB). The result showed that the cleavage rate in single ionomycin was significantly lower than that in Ionomycin+6-DMAP and 6-DMAP+CB (34.38% vs. 69.85% and 72.02%; P &amp;lt; 0.05). However, the cleavage rates and blastocyst rates had no significant difference after in vitro culture (P &amp;gt; 0.05). When the cloned embryos were co-cultured with fetal mouse fibroblast monolayer, the blastocyst development rate increased. The reconstructed embryos were equilibrated 1–3 h, 3–6 h, and 6–9 h after fusion, and then activation was undertaken by ionomycin+6-DMAP. We found that the cleavage rates had no significant difference during 1–3 h and 3–6 h (72.58% vs. 72.97%; P &amp;gt; 0.05), but both were significantly higher than during 6–9 h (64.40%) (P &amp;lt; 0.05). A total of 491 reconstructed embryos were surgically transferred into 37 recipient surrogates, Huanghuai white goats with natural estrus. One of those who were treated with hCG after transfer was pregnant and gave birth to a live kid on 153 days. The lamb died accidentally 8 h after birth. The cloned offspring was then dissected and proved well in all organs. Staining of paraffin tissue slices of the viscera suggested that the organs developed well. Microsatellite analysis indicated that the lamb was derived from the somatic cell donor doe genetically.

Interspecies nuclear transfer reveals that demethylation of specific repetitive sequences is determined by recipient ooplasm but not by donor intrinsic property in cloned embryos

DNA methylation/demethylation of donor genomes in recipient ooplasm after nuclear transfer occurs in a species-specific way. In cloned rabbit and bovine embryos, repetitive sequences maintain the donor-type methylation status, but typical demethylation of repetitive sequences takes place in cloned porcine embryos. To clarify whether the demethylation is controlled by donor nucleus intrinsic property or by recipient ooplasm, we used interspecies somatic cell nuclear transfer (iSCNT) model to examine the methylation status of repetitive sequences in pig-to-rabbit and rabbit-to-pig interspecies embryos. We found that no demethylation of pig repetitive sequences was observed in pig-to-rabbit iSCNT embryos, while the examined rabbit repetitive sequence Rsat IIE was demethylated in rabbit-to-pig iSCNT embryos. These results indicate that demethylation of donor repetitive sequences is determined by ooplasm but not by donor intrinsic property and that ooplasm from different species have different capabilities to demethylate genes.

Development of Interspecies Cloned Embryos in Yak and Dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat–mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig–mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.

28PREGNANCIES RESULTED FROM GOAT NT EMBRYOS PRODUCED BY FUSING COUPLETS IN THE PRESENCE OF LECTIN

The procedure of nuclear transfer (NT) using somatic cells remains inefficient partly due to low fusion rates between donor cells and recipient ooplasm. Lectin is a glycoprotein which specifically binds to carbohydrates to induce a tight contact of membrane to membrane (Booth et al., 2001 Cloning and Stem Cells, 3, 139–159). The purpose of this study was to examine the fusion rates and developmental competence of NT embryos following pre-incubation of couplets in medium containing lectin prior to electrical pulsing. Oocytes were collected by laparoscopic ovum pick-up from hormonally primed goats or by aspiration from culled goat ovaries, and cultured for maturation at 38.5°C, 5% CO2. At approximately 24h after the onset of IVM, the cumulus cells were stripped off by brief vortexing in medium containing 0.2% hyaluronidase. Oocytes with first polar bodies were selected for NT. Successful enucleation was confirmed by the absence of MII chromosomes in ooplasm by means of brief exposure of the Hoechst 33342-stained oocytes to UV light. Three cumulus-granulosa cell lines from transgenic goats were used as donor cells. They were cultured to confluency in DMEM+20% FCS for 6 days prior to NT. Individual donor cells were transferred into the perivitelline space of the enucleated oocytes. Couplets were incubated for 15 minutes in TCM199+10% FCS containing 75 or 150μgmL−1 lectin (L-9132 Sigma, St.Louis, MO, USA) prior to being subjected to electrical pulsing (lectin treatment) with one DC pulse at 2.4kV/cm (1st pulsing). The fusion rate was determined 40–60 minutes after the 1st electric pulsing. Non-fused couplets were exposed to a 2nd pulsing. Approximately 30 minutes later, non-fused couplets were exposed to a 3rd pulsing. Couplets without the lectin treatment served as controls. Reconstructed embryos were activated with 5μM ionomycin followed by 5h of incubation in 10μgmL−1 cycloheximide with 7.5μgmL−1 cytochalasin B. A group of 10 to 13 embryos was transferred into a recipient after 12 to 14h of culture in G1.3. Statistical analysis was performed using the χ2 analysis. Results are shown in the following table. This study demonstrated that fusion rate could be improved by pre-incubating couplets in the medium containing 150μgmL−1 lectin prior to electrical pulsing and the embryos derived from the lectin treatment could establish the early pregnancies.

Mitochondria in human offspring derived from ooplasmic transplantation.

Ooplasmic transfer from fertile donor oocytes into potentially compromised recipient patient oocytes has led to the birth of nearly 30 babies worldwide. Cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial (mt) heteroplasmy (both recipient and donor mtDNA) in offspring. This investigation traced the mitochondrial donor population both during the ooplasmic transfer technique and in the bloods of two 1 year old children using mtDNA fingerprinting. Donor ooplasm stained for active mitochondria was transferred into recipient ooplasm and the mitochondria were visualized by confocal microscopy after the microinjection procedure and fertilization. Heteroplasmy was found in the blood from each of the children. This report is the first case of human germline genetic modification resulting in normal healthy children.

Studies on Nuclear Transfer in Mice

The present study was conducted to establish a cloning system by nuclear transfer technique in mice in which nuclear transfer into 2-cell emrbyos and Metaphase II oocytes were examined and the developmental property of the reconstituted eggs was investigated in vitro and in vitro. The results were summerized as follows. 1) By transfer of a single nucleus from 2-and 4-cell embryos into one of blastomeres of the enucleated 2-cell embryo, identical triplet and twin mice were produced after in vitro culture and transfer to recipients.2) In the nuclear transfer into enucleated oocytes, the chromosomes of oocytes at telophase of the first meiotic division were successfully removed using standard micromanipulation and a differential interference micros-copy. The enucleated oocytes (ooplasts) wewe able to use as recipient ooplasm for nuclear transfer after culture for 4-6h in vitro.3) When the ooplasts received a nucleus from the late 2-cell embryos, 23% developed to blastocysts in vitro, and the offspring were obtained after transfer to recipients. Transfer of a inner cell mass (ICM) cell and a thymocyte from fetuses and new born mice into the ooplasts also resulted in development into blastocyst stage. 4) It was shown that the timing of fusion of donor nucleus with a ooplast and activation of it is essential factor for regulation of the remodelling of the donor nucleus in nuclear transferred oocytes. This study shows the possibility that not only early embryonic nucleui but also more advanced nuclei are reprogrammed when they are transferred into the ooplast in mice.