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Biomedical subjects

A Trounson

Publications and source records attributed to A Trounson.

At least 19 recordsLinked to original sources
PubMed2005

Human embryonic stem cell derivation and directed differentiation.

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine.

Cell Differentiation↗
PubMed2005

Human embryonic stem cells and their spontaneous differentiation.

Human embryonic stem cells (hESCs) usually grow in saucer-shaped colonies with thickened rims and can form spherical human embryoid bodies (hEBs) under non-adherent conditions. A problem associated with ES cell culture is the spontaneous differentiation of cells into a variety of cell types representing all three germ layers, which is evident in both hESC colonies and hEBs. This presentation deals with the precise origins of hESCs and their spontaneous differentiation in vitro. We have used advanced digital microscopy, including transmission electron microscopy (TEM) to define the fine structure of these cells. We present images of undifferentiated hESCs and their spontaneous differentiation into basic embryonic cell types such as nerve, muscle, connective tissue, epithelium, and digestive tract progenitors, representing all three primary germ layers: embryonic ectoderm, mesoderm and endoderm. It appears that hESCs work in concert and interact with one another, as in tissue formation of the embryo. Our fine structural observations agree mostly with those of the Thomson group. Digital microscopy of plastic sections and TEM are invaluable tools in the precise characterization of cells forming these tissues and a combined study with immunofluorescent markers is most desirable.

Cell Communication↗
PubMed2003

In vitro maturation of oocytes from non-stimulated common wombats.

Assisted reproductive techniques, such as in vitro oocyte maturation in conjunction with in vitro fertilisation, may be used as a tool to manipulate reproduction. Using the common wombat as a model for the critically endangered northern hairy-nosed wombat, the present study examined whether oocyte maturation could be achieved under field conditions. At the time of collection, no oocytes were at the metaphase II (MII) stage (0/42). After 60 h culture using the submarine incubation system, 34% of oocytes (24/70) matured to telophase/MII, as indicated by the presence of a polar body. The proportion of oocytes that reached MII was higher for oocytes collected from follicles >2 mm in diameter compared with follicles <2 mm (40% v. 22%, respectively). The presence of cumulus cells alone did not influence the maturation potential. Oocytes without cumulus cells collected from follicles >2 mm in diameter had the highest maturation rate (58%). Maturation was not affected by the reproductive status of the common wombat or a delay of up to 5 h before oocyte collection. In conclusion, the present study demonstrated that oocytes collected from non-stimulated common wombats can mature to MII in culture.

Animals↗
PubMed2003

Effect of ovariectomy and graft position on cryopreserved common wombat (Vombatus ursinus) ovarian tissue following xenografting to nude mice.

Ovarian tissue xenografting may be applied to increase the population size of rare or endangered animals. However, optimal grafting conditions, such as graft position and recipient hormonal status, are yet to be established. The present study, using common wombat ovarian tissue, showed that development of xenografted ovarian tissue to the antral follicle stage can be achieved irrespective of graft position. However, increased graft recovery rates and follicle survival were evident after grafting under the kidney capsule compared with grafting to subcutaneous sites. No increase in follicle development was observed after placing grafts both under the kidney capsule and subcutaneously in the one recipient compared with grafts placed under the kidney capsule alone or subcutaneously alone. Removal of the recipient's own ovaries at the time of grafting accelerated graft follicle development, with antral follicles seen by Week 12 after grafting compared with by Week 16 in recipients that retained their own ovaries. More oocytes were collected from xenograft recipients receiving hormonal stimulation before collection compared with non-stimulated recipients. No oocytes were mature (extruded a polar body) at the time of collection or after a subsequent period of in vitro maturation. This is the first study to demonstrate that antral follicle development can occur and oocytes can be collected from xenografted common wombat ovarian tissue.

Animals↗
PubMed2003

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos.

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.

Animals↗
PubMed2003

Morphological assessment of preimplantation embryo quality in cattle.

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.

Animals↗
PubMed2001

Nuclear transfer of adult and genetically modified fetal cells of the rat.

The present study examines the handling, activation, and micromanipulation of rat eggs in an attempt to produce live young using nuclear transfer (NT) of adult and genetically modified rat fetal cells. Mature rat eggs cultured in calcium-free medium showed reduced rates (24%) of chromosomal dispersion ("spontaneous activation" characteristic of this species) compared with eggs cultured in calcium-containing medium (47%), but failed to survive micromanipulation procedures. High rates of parthenogenetic cleavage were obtained with chemical activation using ethanol/cycloheximide (65%) compared with other standard chemical activation methods (4-28%). This type of activation was also effective in reestablishing cleavage capability (19-71%), in a time-dependent manner, of spontaneously activated eggs arrested at a second prophase-like state. At most, two of four tested micromanipulation procedures were effective in producing NT embryos capable of morula or blastocyst development (14-16%) in vivo following transfer to mouse oviducts. NT blastocysts produced from cumulus cells and transfected rat fetal fibroblasts appeared morphologically and karyotypically normal (2n = 42). Nocodazole-assisted metaphase enucleation and piezoelectric-assisted donor cell injection produced significant and equivocal effects on survival and cleavage rates of reconstructed embryos but failed to significantly improve in vivo morula/blastocyst development rates (16-28%) compared with unassisted micromanipulation (16%). Live births have not yet been obtained from early cleavage stage embryos (n = 269) transferred to pseudopregnant recipient rat oviducts. Improvements in reconstituted NT embryo culture and transfer are required for these methods to be an effective means of transgenic rat production.

Animals↗
PubMed2001

Nuclear transfer in human medicine and animal breeding.

Cloning has a number of potential applications in human medicine and animal breeding, but the efficiency of production of developmentally competent embryos and healthy animal offspring needs to be improved. The primary deficiency appears to be incomplete or abnormal nuclear reprogramming after nuclear transfer, and it is hypothesized that epigenetic regulators of transcription cannot always be converted to the embryonic pattern and this leads to implantation failure, gestational abnormalities and poor health of offspring. Research needs to be focused on this aspect of development for nuclear transfer embryos. However, there is a gradual demand for commercial application for cloning in animal production, transgenesis and animal biopharmaceuticals, particularly in milk. These applications will expand in the near future. There is little application of nuclear transfer in human medicine at present, but important applications may be demonstrated for prevention of the inheritance of mitochondrial mutations, prevention of age-related aneuploidis in women, artificial gametes for sterile individuals or couples and for therapeutic cloning to enable cell and gene therapies.

Animal Husbandry↗
PubMed2001

Cell synchronization for the purposes of nuclear transfer in the bovine.

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1. Cells synchronized in G0, M, and G1 phase were transferred to enucleated bovine MII oocytes by direct injection using the Piezo-Drill microinjector. NT oocytes were artificially activated following injection. Cells at the M phase were also transferred to enucleated oocytes after artificial activation. Cells induced into quiescence by serum starvation and unsynchronized donor cells produced the highest rates of development to the morula/blastocyst stage (20% and 18%, respectively). Development to blastocyst was significantly higher in parthenogenetic controls compared to NT embryos. The transfer of M phase nuclei to MII cytoplasts was not associated with high development to the blastocyst stage. Nevertheless, determining the viability of these embryos requires transfer to recipient animals and assessment of in vivo development.

Animals↗
PubMed2001

Maturation of human oocytes in vitro and their developmental competence.

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Animals↗
PubMed2000

Fine structure of human oogonia in the foetal ovary.

Foetal ovarian tissue is now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. This study examines the ultrastructure of oogonia at 13-15 weeks of gestation, which could be used as a control for culture and freezing of foetal ovaries. Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to dense follicle cells, which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts - beginning of folliculogenesis. Nuclei of oogonia contain one to three highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum (RER) form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristate presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The most remarkable feature of oogonia is that they have typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material, which nucleate microtubules, characteristic of functional centrosomes organizing the cytoskeleton. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced, while the paternal is dominant. Centrioles are most likely involved in mitosis of oogonia.

Centrioles↗
PubMed2000

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Animals↗
PubMed2000

Developmental competence of nuclear transfer cow oocytes after direct injection of fetal fibroblast nuclei.

We have examined the in vitro and in vivo development of cloned embryos produced by incorporation of fetal fibroblast into in vitro matured and enucleated cow oocytes by direct injection and by fusion. For injection, nuclei were either mechanically isolated using the microinjection needle or chemically isolated by treatment with NP-40 lysis buffer. Fetal fibroblasts were serum starved and treated with calcium ionophore before injection to induce chromatin condensation. A range of 8% to 16% of successfully injected oocytes developed to blastocysts in culture and a total of nine pregnancies resulted from transfer of cloned embryos produced by this method. Nuclear transfer by fusion resulted in 22% development to blastocysts. Unlike in mice, the embryos derived from injection did not result in viable pregnancies, which may suggest species differences. All pregnancies were terminated after 45 to 150 days from transfer. Two pregnancies resulted from transfer of cloned embryos obtained by fusion which produced two healthy female calves. The study proposes an alternative method for the production of cow cloned embryos. Further research, however, is required to optimize bovine cloning by injection.

Abortion, Veterinary↗
PubMed2000

Human embryonic stem cells.

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

Animals↗
PubMed1999

Inhibition of bovine sperm-oocyte fusion by a monoclonal antibody recognising the TEC-2 epitope on bovine oocytes.

The TEC-2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC-2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC-02. Incubating oocytes with the TEC-02 antibody prior to fertilization inhibited cleavage in a dose-dependent manner-the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC-02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC-02 antibody had a 7.5 +/- 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 +/- 5.8% of sperm-oocyte fusions and release of cortical granules. The block to sperm-oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC-2 epitope sites involved in the fusion process. In conclusion, the TEC-2 epitope seems to be involved in sperm-oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization.

Animals↗
PubMed1999

Inhibition of bovine sperm-oocyte fusion by the carbohydrate GalNAc.

The TEC-2 epitope is a carbohydrate located on the plasma membrane (oolemma) of the oocyte and appears to be involved in bovine sperm-oolemma fusion. The carbohydrates N-acetylgalactosamine (GalNAc) and galactose are part of the TEC-2 epitope and this study investigated the involvement of these carbohydrates during bovine fertilization. Gametes were exposed to the carbohydrates GalNAc, galactose, and fructose, and the lectins DBA and Con A to determine whether there was an effect on fertilization. The DBA lectin recognizes the carbohydrate GalNAc, whereas Con A recognizes the carbohydrates glucose and mannose. Oocytes pretreated with the DBA lectin prior to fertilization showed a reduction in cleavage corresponding to an increase in lectin concentrations. There was a significant increase in sperm-oolemma binding although fusion was inhibited. Oocytes exposed to GalNAc prior to sperm insemination had no effect on fertilization, however, sperm pretreatment with the carbohydrate caused inhibition of fertilization, with a reduction in cleavage rates as the GalNAc concentration increased. There was also a significant decrease in sperm-oolemma fusion and a significant increase in sperm-oolemma binding. When gametes were exposed to GalNAc at the time of fertilization a similar response to that seen with sperm pretreatment was observed. The carbohydrates galactose and fructose and the lectin Con A did not affect fertilization. In conclusion, the carbohydrate GalNAc, which is associated with the TEC-2 epitope, has a specific role during bovine sperm-oolemma fusion. This study also suggests that there is a carbohydrate-binding molecule on the sperm that binds GalNAc.

Acetylgalactosamine↗