Somatic cell cloning without micromanipulators.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively.

DNA fingerprinting of sister blastomeres from human IVF embryos.

Background: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis.

Methods: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software.

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations.

Comparison of gene transcription in cloned bovine embryos produced by different nuclear transfer techniques.

The efficiency of animal production using cloning technology is still relatively low and research to determine a more efficient nuclear transfer procedure is ongoing. One approach which may be informative in assessing the viability of nuclear transfer embryos is the analysis of embryonic gene expression. Using RT-PCR techniques we have previously detected the aberrant expression of FGF4, FGFr2 and IL6 in a significant proportion of bovine granulosa cell-derived nuclear transfer embryos, which correlated with a limited developmental potential in vivo.

Sex ratio and birth weights of infants born as a result of blastocyst transfers compared with early cleavage stage embryo transfers.

Objective: To analyze the birth weights and sex ratio of infants born as a result of blastocyst transfer and compare them with data resulting from the transfer of early-cleavage stage embryos.

Design: Retrospective analysis.

Setting: Monash IVF (private in vitro fertilization clinic).

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.

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%).

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.

A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen.

Double packaging, in which an inner straw containing the specimen is inserted into an outer, larger straw (here termed 'straw-in-straw') to prevent the inner straw from coming into direct contact with liquid nitrogen provides a simple strategy for reducing or eliminating the potential contamination risk associated with storage in liquid nitrogen. This approach has in the past been used in conjunction with cryopreservation by slow cooling, but has not previously been tested for use throughout an entire rapid cooling and warming procedure. This study determined whether keeping the straw containing the embryos inside a second protecting container throughout the cryopreservation and storage protocol would compromise embryo viability.

Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting.

Ovarian grafting provides a strategy for clinical infertility treatment and is starting to be used in conjunction with ovarian tissue storage for patients at risk of early ovarian failure. As patients are starting to return for their frozen stored tissue we need to ascertain how to maximise follicle survival when this tissue is grafted back to the patient. For research purposes ovarian tissue is commonly grafted to the kidney capsule as the rich capillary bed at this site favours rapid graft revascularization.

Vitrification of the oocytes and embryos of domestic animals.

After the first successful application of vitrification for embryo cryopreservation 15 years ago, a rapid application of the method in domestic animal embryology was presumed. However, although the advantages of vitrification (simplicity, cost efficiency, speed of the procedure) were widely acknowledged, its use has been mainly restricted to experimental studies. For commercial embryo transfer purposes, the traditional slow-rate or equilibrium freezing has been used.

Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications.

Ovarian tissue storage has several potentially very valuable clinical applications, including the management of young female patients that are at risk of premature menopause. Ovarian tissue collection, used alone or in combination with oocyte and embryo cryopreservation, may help these patients safeguard their own future fertility. All available evidence from animal studies indicates that grafting of frozen ovarian tissue should be feasible in the human.

Fundamental cryobiology of mammalian oocytes and ovarian tissue.

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible.

Current and future prospects for xenotransplantation.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g.

Reprogramming cattle somatic cells by isolated nuclear injection.

The development of a somatic cell nuclear transfer procedure for the production of blastocyst stage cattle embryos is described. Bovine fetal fibroblasts were used for fusion experiments with surgically enucleated oocytes (cytoplasts) following the establishment of optimal parameters for electrofusion from isofusion contours. Fusion rates were increased by decreasing size of the cytoplasts used but cleavage was decreased by decreasing size of the cytoplast used (quarter, half and whole cytoplasts).

Novel method for demonstrating nuclear contribution in mouse nuclear transfer.

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring.

Birth following vitrification of a small number of human oocytes: case report.

We report the birth of a healthy baby girl at 37 weeks gestation to a 47 year old recipient, after vitrification of mature oocytes from four in-vitro fertilization (IVF) patients. A total of 17 oocytes was vitrified in 1-2 microl of ethylene glycol (40%) and 0.6 mol/l sucrose (20.

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.

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.

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.

Temporal and differential effects of amino acids on bovine embryo development in culture.

The aim of the study was to determine the amino acid requirements of the in vitro-produced bovine embryo as it develops from the zygote to the blastocyst, using a two-step culture system. When added to synthetic oviduct fluid (SOF) for the first 72-h culture, Eagle's nonessential amino acids and glutamine (NeGln) significantly increased development to the 8- to 16-cell stage (Day 4 postinsemination [pi]) and subsequent blastocyst development (Day 7 pi). Glutamine alone during the first 72-h culture did not stimulate development to the 8- to 16-cell stage (p > 0.

Comparison of the expression of specific cell surface epitopes on in vitro fertilized and parthenogenetic bovine embryos.

Parthenogenetic embryos, which are produced by the spontaneous or artificial stimulation of the oocyte, partially develop in the complete absence of the male gamete but fail to produce live young in many mammalian species. The identification of developmentally regulated molecules on the cell surface of embryos has implicated their possible role in cell interactions during embryogenesis and differentiation. In this study the expression patterns of four stage-specific cell surface antigenic determinants (TEC-1, -2, -3, and -4) were investigated in both parthenogenetic and in vitro fertilized bovine embryos.

Metabolism of glucose, pyruvate, and glutamine during the maturation of oocytes derived from pre-pubertal and adult cows.

The aim of the study was to compare the energy metabolism of oocytes from pre-pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre-pubertal oocytes. The metabolism of [5-(3)H] glucose, [2-(14)C] pyruvate, and [G-(3)H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre-pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr.

In vitro development and nutrient uptake by embryos derived from oocytes of pre-pubertal and adult cows.

The present study compared the developmental potential and uptake of nutrients by embryos from pre-pubertal and adult cows. Oocytes retrieved from ovaries of 5 to 7 month old calves and adult cows were matured and fertilized in vitro. Embryos were cultured in SOFaa to the blastocyst stage (7 days post-insemination).