Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

Amino acid uptake and protein synthesis in preimplanatation mouse embryos

Amino acid uptake and cycloheximide inhibitable incorporation into protein are demonstrable in follicular ova, unfertilized eggs, and in mouse embryos ranging from the 1-cell to the late blastocyst stages. The rates of uptake and incorporation are low and relatively constant until the early blastocyst (day 3) stage of development when they increase 3- to 9-fold. The rate of uptake continues to increase during the fourth day (late blastocyst stage) of development, but, despite embryonic growth, incorporation into protein remains constant. By exposing embryos to leucine and lysine at different concentrations, it is possible to dissociate the processes of uptake and incorporation into protein from one another and to use the latter as a measure of protein synthesis. Taking the number of embryonic cells into account, it is postulated that the period between 8- to 16-cell stage (day 2) and the early blastocyst stage is the only one in which the synthesis of major amounts of protein based on new messenger RNA synthesis is occurring.Leucine and lysine uptake take place by a facilitated process, although lysine transport is not readily saturated. Inhibitors of energy metabolism do not significantly affect amino acid uptake, but they do inhibit protein synthesis. However, since the rate of transport is highly temperature sensitive (Q₁₀ ? 3) and leucine is accumulated against a concentration gradient, active amino acid transport appears to be present.     

Differential expression pattern of key regulatory developmental genes in pre-implant zona free cloned vs in vitro fertilized goat embryos

The success of Somatic cell nuclear transfer (SCNT) primarily depends on the extent of reprogramming of donor cells genome. The error of reprogramming may lead to inappropriate expression of embryonic genes at any stage of development. Under the present study the relative expression of different genes related to pluripotency (Oct-4 and Nanog), growth factors (IGF-2 and IGF-2R) and DNA methyltransferase gene (Dnmt-1) was evaluated in SCNT embryos at 8–16 cells, morula and blastocyst stages as compared to IVF group. In SCNT, significantly higher degree of relative expression was observed for Oct-4 in morula (1.41) and blastocysts (1.14) as compared to 8–16 cells (referral stage) whereas in IVF, a lower expression was observed at morula (0.82) stage. The expression of Nanog in SCNT embryos was increased significantly in morula (2.23) and decreased subsequently in blastocyst (0.56), whereas it was increased significantly from 8 to 16 cells to morula (1.62) and blastocyst (4.5) of IVF group. The IGF-2 and IGF-2R showed significantly higher expression rates in morula and blastocysts of SCNT (6.56, 5.90 and 1.11, 1.4) and IVF (8.69, 8.25 and 2.96, 3.91) embryos, respectively as compared to referral stage. The expression of Dnmt-1 was significantly higher in SCNT morula (1.29) and blastocyst (1.15) however in IVF, it was similar in 8–16 cells stage and morula but, higher in blastocyst (1.58). The dissimilar pattern of gene expression of SCNT might be a consequence of incomplete reprogramming of donor nucleus which resulted into lower blastocyst rate of SCNT as compared to IVF embryos.     

The giant panda ( Ailuropoda melanoleuca ) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg. Project supported by the 1998 Special Fund of the Chinese Academy of Sciences (KY95-J1-318) and a 1999 Special Fund from the Ministry of Science and Technology. Instruments were dominated by Mr. Shum Yam Wa, Heal Force Development Ltd.     

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.     

Comparative embryology and mammalian cloning

A hypothesis has been advanced that logically combines “contradictory” facts concerning the early mammalian development and shows a natural relationship between the embryos developing from a fertilized ovum and from cells of the inner cell mass of blastocyst. When studying the theoretical questions of cloning, it is necessary to take into consideration the peculiarities of prenatal mammalian ontogeny, which make themselves evident upon comparison with other animals. The absence of yolk in the mammalian ovum defines sharp differences in the early development between mammals and other Amniota. The complete asynchronous cleavage results in the formation of morula followed by blastocyst, which hatches from zona pellucida and is implanted into the uterus tissue. This fact allows us to consider the blastocyst as a mammalian larva, which is fed owing to the maternal organism. It is known that, in the body of a larva (blastocyst), a new embryo develops from some somatic cells. This process is known as polyembryony, which is typical of the development of some parasitic insects. Polyembryony in turn is a variant of somatic embryogenesis, which is a form of asexual reproduction. Thus, the two different embryos, “conceptus” and “embryo proper,” have different origins: the first forms by the sexual way and the second, by the asexual way. Investigation of the mechanisms of somatic embryogenesis in mammals will help us to find conditions necessary for full reprogramming of donor somatic nuclei and provide for successful development of reconstructed embryos.     

Epigenetic regulation and role of LINE-1 retrotransposon in embryogenesis

LINE-1 retrotransposon is the most common mobile genetic element in the genomes of various mammals, including humans. Its genes are represented by the greatest number of copies. For a long time, it has been considered that the presence of LINE-1 in genome reflects the limited ability of cells to eliminate it, and the retrotransposon activity is negative owing to the insertional mutagenesis. In recent years, the increased expression of LINE-1 retrotransposon and the activity of their encoded proteins observed in mammalian cells at different stages of development and, first of all, in early embryogenesis have been discussed in the literature. Is early embryogenesis the stage of development when the organism is more susceptible to the activity of retrotransposons, or does LINE-1 play some positive role in early embryonic development? This review is aimed at classifying the available data on the epigenetic regulation and the role of LINE-1 retrotransposon in embryogenesis of mammals. The link between the mechanisms of regulation of LINE-1 expression and the waves of epigenetic reprogramming is tracked in germ cells, during fertilization, and in blastocyst, as well as during the differentiation of embryonic and extraembryonic tissues.     

Blastocysts produced by nuclear transfer between chicken blastodermal cells and rabbit oocytes

Interspecies nuclear transfer (INT) has been used as an invaluable tool for studying nucleus-cytoplasm interactions; and it may also be a method for rescuing endangered species whose oocytes are difficult to obtain. In the present study, we investigated interaction of the chicken genome with the rabbit oocyte cytoplasm. When chicken blastodermal cells were transferred into the perivitelline space of rabbit oocytes, 79.3% of the couplets were fused and 9.7% of the fused embryos developed to the blastocyst stage. Both M199 and SOF medium were used for culturing chicken-rabbit cloned embryos; embryo development was arrested at the 8-cell stage obtained in SOF medium, while the rates of morulae and blastocysts were 12.1 and 9.7%, respectively, in M199 medium. Polymerase chain reaction (PCR) amplification of nuclear DNA and karyotype analyses confirmed that genetic material of morulae and blastocysts was derived from the chicken donor cells. Analysis mitochondrial constitution of the chicken-rabbit cloned embryos found that mitochondria, from both donor cells and enucleated oocytes, co-existed. Our results suggest that: (1) chicken genome can coordinate with rabbit oocyte cytoplasm in early embryo development; (2) there may be an 8- to 16-cell stage block for the chicken-rabbit cloned embryos when cultured in vitro; (3) mitochondrial DNA from the chicken donor cells was not eliminated until the blastocyst stage in the chicken-rabbit cloned embryos; (4) factors existing in ooplasm for somatic nucleus reprogramming may be highly conservative.     

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1^nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.     

Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.     

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.     

Epiblast isolation by a new four stage method (peeling) from whole bovine cloned blastocysts

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new “peeling” method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin β3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.     

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1–5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (X², P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals. © 1996 Wiley-Liss, Inc.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.     

Analysis of cell lineage in two- and four-cell mouse embryos

Compared with other animals, the embryos of mammals are considered to have a highly regulative mode of development. However, recent studies have provided a strong correlation between the first cleavage plane and the future axis of the blastocyst, but it is still unclear how the early axes of the preimplantation embryo reflect the future body axes that emerge after implantation. We have carried out lineage tracing during mouse embryogenesis using the Cre-loxP system, which allowed us to analyze cell fates over a long period of development. We used a transgenic mouse strain, CAG-CAT-Z as a reporter line. The descendants of the manipulated blastomere heritably express beta-galactosidase. We examined the distribution of descendants of a single blastomere in the 8.5-day embryo after labeling at the two-cell and four-cell stages. The derivatives of one blastomere in the two-cell embryo randomly mix with cells originating from the second blastomere in all cell layers examined. Thus we find cells from different blastomeres intermingled and localized randomly along the body axis. The results of labeling experiments performed in the four-cell stage embryo fall into three categories. In the first, the labeled cells were intermingled with non-labeled cells in a manner similar to that seen after labeling at the two-cell stage. In the second, labeled cells were distributed only in the extra-embryonic ectoderm layers. Finally in the third category, labeled cells were seen only in the embryo proper and the extra-embryonic mesoderm. Manipulated embryos analyzed at the blastocyst stage showed localized distribution of the descendants of a single blastomere. These results suggest that incoherent clonal growth and drastic cell mixing occurs in the early mouse embryo after the blastocyst stage. The first cell specification event, i.e., partitioning cell fate between the inner cell mass and trophectoderm, can occur between the two-cell and four-cell stage, yet the cell fate is not determined.     

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg.     

Patterning of the embryo: the first spatial decisions in the life of a mouse

Although in most species the polarity of the embryo takes its roots from the spatial patterning of the egg, mammals were viewed as an exception. This was because the anteroposterior polarity of the mouse embryo could not be seen until gastrulation, and no developmental cues were known that could define polarity at earlier stages. Why should we now re-consider this view? While mechanisms of axis formation in mammals could, in principle, be unique, the evolutionary conservation of numerous other developmental processes raises the question of why mammals would have evolved a different way or timing of organising their embryonic polarity. Indeed, recent evidence shows that well before the onset of gastrulation, the mouse embryo initiates asymmetric patterns of gene expression in its visceral endoderm. Although this extra-embryonic tissue does not contribute to the body itself, it is involved in axis formation. Other recent work has revealed that spatial distribution of cells in the visceral endoderm can be traced back to polarity present at the blastocyst stage. These insights have raised the possibility that embryonic polarity might also originate early during development of mammalian embryos. Indeed it now appears that there are at least two spatial cues that operate in the mouse egg to shape polarity of the blastocyst. One of these is at the animal pole, which is defined by the site of female meiosis, and another is associated with the position of sperm entry. In this review I discuss these recent findings, which have led to the recognition that mouse embryos initiate development of their polarity at the earliest stages of their life. This novel perspective raises questions about the nature of cellular and molecular mechanisms that could convert developmental cues in the zygote to axes of the blastocyst, and hence into polarity of the post-implantation embryo. It also brings to light the need to understand how such mechanisms could enable early mouse development to be so regulative.     

In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells

The present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at -4 degrees C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at -196 degrees C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.