Embryo Reconstruction Research Articles

Prenatal origins of human intrapulmonary arteries formation and smooth muscle maturation

Recent studies on the morphogenesis of the pulmonary arteries have focused on nonhuman species such as the chick and the mouse. Using immunohistochemical techniques, we have studied 16 lungs from human embryos and fetuses from 28 d of gestation to newborn, using serial sections stained with a panel of antibodies specific for endothelium, smooth muscle, and extracellular matrix proteins. Cell replication was also assessed. Serial reconstruction showed a continuity of circulation between the heart and the capillary plexus from at least 38 d of gestation. The intrapulmonary arteries appeared to be derived from a continuous expansion of the primary capillary plexus that is from within the mesenchyme, by vasculogenesis. The arteries formed by continuous coalescence of endothelial tubes alongside the newly formed airway. Findings were consistent with the pulmonary arterial smooth muscle cells being derived from three sites in a temporally distinct sequence: the earliest from the bronchial smooth muscle, later from the mesenchyme surrounding the arteries, and last from the endothelial cells. Despite their different origins, all smooth muscle cells followed the same sequence of expression of smooth muscle‐specific cytoskeletal proteins with increasing age. The order of appearance of these maturing proteins was from the subendothelial cells outward across the vessel wall and from hilum to periphery. The airways would seem to act as a template for pulmonary artery development. This study provides a framework for studying the signaling mechanisms controlling the various aspects of lung development. The past few years have seen advances in our understanding of pulmonary endothelial and smooth muscle cell phenotype and function, and yet our understanding of the origins of the human pulmonary arteries is still based on the reconstructions of embryos described by Congdon in 1922 (1). Later studies showed that intrapulmonary preacinar airway and arterial branching is complete by 16 wk of gestation (2), but the origins of the capillary plexus and its connection with the pulmonary arteries have remained unclear. A recent study has shown that in the mouse, the pulmonary circulation appears to derive from two sources (3). In that study, using casts and electron microscopy, segmental arteries were shown to develop by angiogenesis from the extrapulmonary circulation, and simultaneously, the peripheral capillary plexus formed by vasculogenesis from the pulmonary mesenchyme. The communication between the two was completed at 13 to 14 d of gestation (3). In the present study, we have studied the development of the capillary plexus in human embryonic and fetal lungs on serial sections, having immunostained the endothelial cells for expression of cluster of differentiation number 31 (CD31) and von Willebrand factor. Newly formed endothelial tubes in the systemic and pulmonary circulation become invested by smooth muscle cells, but the origins of these cells are not certain. In vitro studies indicate that smooth muscle cells may be recruited by the endothelium from the surrounding mesenchyme or arise by transdifferentiation from the endothelium (4). In the systemic circulation of the developing quail embryo, mesenchymal cells associating with the endothelium express smooth muscle‐specific a -actin ( a -SM-actin) and these committed cells then express more smooth muscle cell markers as they mature (5). The phenotype of vascular smooth muscle cells varies, with adult bovine and porcine pulmonary arteries containing several different cell phenotypes, differing in their cytoskeletal composition (6, 7). In the present study, we have explored the hypothesis that the different muscle phenotypes have different developmental origins. We have examined the temporal and spatial pattern of pulmonary arterial smooth muscle maturation by studying the expression of smooth muscle‐specific, contractile, and cytoskeletal proteins using immunohistochemical labeling in the human fetal lung. Cell migration during tissue morphogenesis is regulated in part by the composition of the tissue matrix. Fibronectin is a chemoattractant for mesenchymal cells (8), and in vitro migration of smooth muscle cells is dependent on fibronectin (9). Expression of tenascin mediates cell migration and proliferation (10‐12). Versican is expressed at sites of tissue interaction in the developing fetal mouse where it regulates paths of cell migration (13, 14). Therefore, we also examined the temporal and spatial distribution of these three proteins in relation to the development of the pulmonary arteries.

Cloned pigs produced by nuclear transfer from adult somatic cells.

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

Embryo cloning by nuclear transfer: experiences in sheep

Although technically established in mammalian species more than 15 year ago, the efficiency of embryo cloning by nuclear transfer in terms of offspring production has been invariably low. This article reviews a series of interrelated experiments carried out from 1992 to 1997 regarding the co-ordination of nuclear and cytoplasmic events after embryo reconstruction. The experiments were undertaken at the Istituto Zootecnico e Caseario per la Sardegna (IZCS) and the sheep used belonged to the Sardinian dairy breed. The use of enucleated, pre-activated recipient cytoplasts resulted in an increased frequency of development to blastocyst of embryos reconstructed with unsynchronized blastomere nuclei. This increase in development was due to the absence of chromatin damage and unbalanced ploidy in nuclei transferred after the decay of MPF (Maturation Promoting Factor). The combination of synchronous S-phase cytoplast-karyoplast nuclear transfer together with the reduction of embryo losses from the oviduct of temporary recipients allowed for the first time the production of large numbers of clones of genetically identical lambs, thus bringing nuclear transfer closer to practical application. Finally, the last series of experiments described deals with the establishment of an alternative protocol for embryonic nuclear transfer where the combined use of a chemical activating agent, ionomycin, with a protein kinase inhibitor, 6-Dimethylaminopurine, resulted in the highest development rates to blastocyst stage of metaphase II enucleated oocytes reconstructed with embryonic nuclei described so far.

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically- chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley-Liss, Inc.

Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos.

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blastomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically-chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentall

Reprogramming of Fibroblast Nuclei after Transfer into Bovine Oocytes

Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

3D Computer modeling of human cardiogenesis

We obtained digitized histological serial sections of three human embryos of different stages from the Armed Forces Institute of Pathology from which contours of cardiac structures were digitally traced. Traces were placed in register for the construction of wire frame models using Silicon Graphics Incorporated 3D computer modeling software (Alias/Wavefront) and for surface rendering to demonstrate internal and external developmental anatomy. The models allowed stage-to-stage morphing and physical model construction using stereolithography. These innovations in embryo reconstruction not only facilitate medical education, they also serve as new tools for scientific investigation of cardiogenesis and congenital heart disease.

The use of activated and aged-cooled oocytes as host ooplasts in bovine nuclear transfer

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5 mM/ml inomycin with 2 mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168 h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48 h after activation, 82% of the cleaved oocytes developed to 2–16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.

In-vivo three-dimensional ultrasound reconstructions of embryos and early fetuses

Three-dimensional (3D) imaging of the living human embryo has become possible in the monitoring of embryological development, as described by classic human embryology. We aimed to create 3D images of organs in embryos on early pregnancy. We used a specially developed 7.5 MHz annular array 3D transvaginal probe to examine embryos. We included 34 women at 7-10 weeks' gestation. We measured the crown-rump length (CRL) of the embryos and fetuses and transferred the 3D ultrasound data to an external computer for further processing to calculate volume. The CRLs ranged from 9.3 mm to 39.0 mm. The quality of the images of the embryos and fetuses made it possible to outline in detail the outer contours and the contours of the brain cavities, and the calculated volumes corresponded well to the descriptions from classic human embryology. Our 3D imaging system allowed visualisation of structures of less than 10 mm. Therefore, development and abnormal development of fetuses can be monitored.

Three-dimensional reconstruction of live embryos using robotic macroscope images.

To determine the three-dimensional (3-D) shape of a live embryo is a technically challenging task. We show that reconstructions of live embryos can be done by collecting images from different viewing angles using a robotic macroscope, establishing point correspondences between these views by block matching, and using a new 3-D reconstruction algorithm that accommodates camera positioning errors. The algorithm assumes that the images are orthographic projections of the object and that the camera scaling factors are known. Point positions and camera errors are found simultaneously. Reconstructions of test objects and embryos show that meaningful reconstructions are possible only when camera positioning and alignment errors are accommodated since these errors can be substantial. Reconstructions of early-stage axolotl embryos were made from sets of 33 images. In a typical reconstruction, 781 points, each visible in at least three different views, were used to form 1511 triangles to represent the embryo surface. The resulting reconstruction had a mean radius of error of 0.27 pixels (1.1 microns). Mathematical properties of the reconstruction algorithm are identified and discussed.

Telophase enucleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer.

The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4 degrees C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

PHYSIOLOGICAL “CONSTANTS” FOR PBPK MODELS FOR PREGNANCY

Physiologically based pharmacokinetic (PBPK) models for pregnancy are inherently more complex than conventional PBPK models due to the growth of the maternal and embryo/ fetal tissues. Physiological parameters such as compartmental volumes or flow rates are relatively constant at any particular time during gestation when an acute experiment might be conducted, but vary greatly throughout the course of gestation (e.g., contrast relative fetal weight during the first month of gestation with the ninth month). Maternal physiological parameters change during gestation, depending upon the particular system; for example, cardiac output increases by -50% during human gestation; plasma protein concentration decreases during pregnancy; overall metabolism remains fairly constant. Maternal compartmental volumes may change by 10–30% embryo/fetal volume increases over a billionfold from conception to birth. Data describing these physiological changes in the human are available from the literature. Human embryo/fetal growth can be well described using the Compertz equation. By contrast, very little of these same types of data is available for the laboratory animal. In the rodent there is a dearth of information during organogenesis as to embryo weights, and even less organ or tissue weight or volume data during embryonic or fetal periods. Allometric modeling offers a reasonable choice to extrapolate (approximately) from humans to animals; validation, however, is confined to comparisons with limited data during the late embryonic and fetal periods of development (after gestation d 11 in the rat and mouse). Embryonic weight measurements are limited by the small size of the embryo and the current state of technology. However, the application of the laser scanning confocal microscope (LSCM) to optically section intact embryos offers the capability of precise structural measurements and computer-generated three-dimensional reconstruction of early embryos. Application of these PBPK models of pregnancy in laboratory animal models at teratogenically sensitive periods of development provides exposure values at specific target tissues. These exposures provide fundamentally important data to help design and interpret molecular probe investigations into mechanisms of teratogenesis.

The mouse atlas and graphical gene-expression database

The large amounts of gene-expression data on mouse development are now too extensive to be stored in any format other than that of a database. Furthermore, as this data is intrinsically graphical and as, in the early developmental stages at least, its boundaries do not map directly to those of anatomical tissues, the natural way to store it is in graphical format. We are therefore constructing a database able to handle such graphical gene-expression data by mapping it onto 3-D reconstructions of mouse embryos whose tissues have been delineated. This article reviews the progress that has been made in this project and describes its two major components, CD-ROMs of the 3-D reconstructions to be held on the user’s computer and a gene-expression database that will be maintained at a host site, the two being linked over the internet by a complex Java-based interface for submitting data and querying the database.

Embryo cloning in sheep: Work in progress

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 μg/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3–6 kg), while the weight for females was normal.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of 6-dimethylaminopurine.

The methods used to achieve blastomere cell cycle synchronization in embryos used as nuclear donors during embryo reconstruction have been largely unsuccessful. The aim of this study was to determine the reliability of 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor, to half and to synchronize blastomere division in cleavage stage bovine embryos. A second goal was to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h after insemination were treated with several concentrations of 6-DMAP for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, histone H1 and scored for blastocyst formation and for hatching rate. They were subsequently fixed and the number of nuclei counted. Complete arrest of cell division was observed at concentrations of 3 mmol 6-DMAP l-1 and above. At these concentrations, interphase nuclei in arrest were noticeably larger compared with interphase nuclei of eight-cell control embryos. Removal from 6-DMAP led to release from cleavage arrest and was followed by synchronized mitosis, histone H1 kinase deactivation and re-entry into interphase within 4-5 h. Twenty-nine per cent of interphase nuclei were synthesizing DNA at the end of the 12 h treatment as indicated by BrdU analysis. At 2 h after removal from 6-DMAP, an abrupt decrease to 9% BrdU-positive nuclei was observed followed by an increase to 39% by 6 h and a decrease to 28% at 10 h. The ability of treated embryos to reach the blastocyst stage in vitro and the number of cells per blastocyst were reduced. These results indicate that 6-DMAP can reversibly arrest and synchronize cleavage to the fifth cell cycle in eight-cell bovine embryos. Although a decrease was observed in the proportion of blastocysts obtained after treatment, it is concluded that 6-DMAP is a useful tool for synchronization studies requiring donor nuclei at metaphase before fusion to recipient oocyte.

The behaviour of mitochondria and cell integration during somatic hybridisation of sister blastomeres of the 2-cell mouse embryo.

The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage. At the time corresponding to the G2/M-phase of the cell cycle the blastomeres fused in only 8% of cases. Vital staining of 2-cell embryos by rhodamine 123 showed that the mitochondria were dispersed throughout the cytoplasm with a ring-like (around the nucleus) or spot-like (over the metaphase plate) concentration in the centre of each blastomere. At the periphery of blastomeres the mitochondrial content was low. The behaviour of the mitochondria reflected the subsequent events of structural and functional integration of the sister blastomeres under induced fusion: a discernible boundary between partners during 30 min after electrofusion or 1 h after fusion with polyethylene glycol; movement of the two 'rings' to the centre of the blastomere fusion products (BFP) to form one large bright 'spot' over the common metaphase plate; mitochondria outlining the shape of the spindle and connection between sister blastomeres until completion of the first mitosis of BFP. The data obtained suggest that fusion of the blastomeres does not lead to extensive changes in the hybrid cytoplasm and integration of nuclear material is taking place only at metaphase stage. Cytogenetic examination of BFP at the 2-cell stage confirmed reconstruction of the tetraploid embryos and found that sister blastomeres of such embryos could asynchronously enter the next cleavage division similarly to normal diploid 2-cell embryos.

Computer-aided 3-D reconstruction of serially sectioned mouse embryos: its use in integrating anatomical organization.

This paper reviews recent work on a project that uses a computer-aided approach for making 3-D reconstructions of serially sectioned mouse embryos (the digital mouse). The captured images are aligned using a warping program so that almost perfect alignment of adjacent sections is achieved with minimal deformation. The sections that are viewed on the computer screen are in fact computer-generated grey-level images with a resolution of about 10 microm. The reconstructed embryo may then be resectioned in any plane to simulate as near as possible an exact match on the computer screen to the viewer's own material. Individual anatomical domains may then be painted in different colors, and these domains may be selected by querying the textual database containing anatomical and other information. Further, it is now possible to generate 3-D images of individual anatomically-discrete components or related sets of components of a particular system in isolation from the rest of the embryo, or, if required, against a 'ghost-like' image of the intact embryo, or specific parts of an embryo. In the article, examples are given of the use of the system in interpreting the vascular, gut and paraxial mesoderm systems, while both the advantages and disadvantages of this approach are also discussed. The eventual aim will be to provide 3-D reconstructions of mouse embryos from fertilization up to 14 days postcoitum of development. When completed, this project will allow the accurate spatial mapping of gene-expression and cell lineage data onto the digital Atlas of normal mouse embryonic development.

Cell cycle co-ordination in embryo cloning by nuclear transfer.

Exciting new opportunities in embryo cloning have been made possible by recent studies on the interaction of the donor nucleus with the recipient cytoplasm after embryo reconstruction. This article reviews information regarding the co-ordination of nuclear and cytoplasmic events during embryo reconstruction, in particular the direct and indirect effects of maturation/ meiosis/mitosis-promoting factor (MPF), upon the transferred nucleus. This will be discussed in relation to DNA replication, the maintenance of correct ploidy, the occurrence of chromosomal abnormalities and development of reconstructed embryos. Although this review is primarily concerned with the reconstruction of mammalian embryos, specific examples from amphibians will also be cited.

Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes.

The timing of pronuclear formation and the initiation and duration of the DNA synthetic period (S-phase) were determined during the first cell cycle of electrically activated ovine oocytes matured in vivo. Reconstructed embryos were produced by electro-fusion-mediated nuclear transfer of unsynchronized single blastomeres. These were derived from embryos produced in vivo at the 16-cell stage (Day 4) and transferred to enucleated metaphase II oocytes at the time of activation or to enucleated activated oocytes during early, mid, and late stages of the presumptive S-phase. The frequency of development to blastocyst was greatest in embryos reconstructed during the presumptive S-phase of enucleated activated oocytes than in embryos reconstructed at the time of activation (mean 55.4% vs. 21.3%). No significant differences were observed when embryos were reconstructed during early, mid, or late stages of the presumptive S-phase (61.3%, 45.7%, and 57.7%, respectively). The results indicate that the use of enucleated activated oocytes as cytoplasts for embryo reconstruction can increase the frequency of development to blastocyst of embryos reconstructed from unsynchronized donor blastomeres.

Computer-Assisted Reconstruction of Vertebrate Embryos from Serial Histological Sections

Computer-Assisted Reconstruction of Vertebrate Embryos from Serial Histological Sections