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I. WILMUT and C. POLGE

Summary.

Three experiments have been carried out to investigate the mechanism by which glycerol reduces the fertilizing capacity of boar spermatozoa during storage, and any relationship between this effect and the relatively poor conception rates obtained with deep-frozen boar semen.

The loss of fertilizing capacity in the presence of glycerol was shown to be temperature-dependent, Fertilizing capacity was significantly reduced after 6 hr of incubation at 20°C in the presence of 5 or 10% glycerol, although there was no detectable change in motility or morphology of the spermatozoa. By contrast, there was no effect on fertilizing ability after storage for 6 hr at 5°C or for 30 min at 20°C in the presence of these concentrations of glycerol. Removal of the seminal plasma and a large proportion of the free cytoplasmic droplets did not prevent the reduction in fertilizing capacity. When semen was frozen and thawed, attempts to minimize the exposure of spermatozoa to glycerol did not lead to fertilization when the thawed semen was inseminated through the cervix. It is suggested that at ambient temperature glycerol causes damage to boar spermatozoa which is exacerbated by the stress of freezing and thawing.

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I. Wilmut and D. I. Sales

Summary. Sheep embryos which were transferred to recipients in oestrus 3 days before or 3 days after the donors were unable to implant even when a synchronously transferred embryo was developing successfully in the same uterus. Embryos which had spent 3 days in an advanced recipient were transferred to a recipient synchronous with the original donor. Embryos first transferred on Day 3 were slightly accelerated in their development, but retained the ability to implant normally in the 2nd recipient. By contrast, embryos first transferred on Day 6 were markedly stimulated and less able to implant in the second recipient.

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L. C. Smith and I. Wilmut

Nuclear–cytoplasmic interactions during the second cell cycle of mouse embryos were examined by assessing the timing of cleavage in reconstituted two-cell embryos and their ability to develop further to the blastocyst stage in vitro. Nuclear transplantations were performed either within or across the cell cycle and at different stages of the cell cycle to assess the effect of 'asynchrony' on development. In most cases, cleavage occurred at an intermediate time between nuclear and cytoplasmic controls indicating an interaction in their mechanisms for controlling the timing of cleavage. Early nuclei extended the cleavage timing of late cytoplasm for a short period, possibly to allow for completion of DNA synthesis, while early cytoplasm delayed the expected cleavage time of late nuclei, possibly to enable proper maturation of cytoplasmic components. However, a block of cleavage was observed in most across cell cycle transplantations and also when fusing early two-cell karyoplasts to enucleated late two-cell blastomeres. It is suggested that this incompatibility is caused by major changes in the transcriptional status of donor and recipient cells. Although the development of reconstituted embryos to the blastocyst stage was clearly affected by cell cycle 'asynchrony' in within cell cycle transplantations, independent effects of cytoplast stage and, to a lesser extent, of karyoplast cell cycle stage were predominant in transplantations using eight-cell karyoplasts.

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LE Young, KD Sinclair and I Wilmut

Bovine and ovine embryos exposed to a variety of unusual environments prior to the blastocyst stage have resulted in the development of unusually large offspring which can also exhibit a number of organ defects. In these animals, the increased incidence of difficult parturition and of fetal and neonatal losses has limited the large-scale use of in vitro embryo production technologies commonly used in humans and other species. Four different situations have been identified that result in the syndrome: in vitro embryo culture, asynchronous embryo transfer into an advanced uterine environment, nuclear transfer and maternal exposure to excessively high urea diets. However, programming of the syndrome by all of these situations is unpredictable and not all of the symptoms described have been observed universally. Neither the environmental factors inducing the large offspring syndrome nor the mechanisms of perturbation occurring in the early embryo and manifesting themselves in the fetus have been identified.

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I. Wilmut, D. I. Sales and C. J. Ashworth

Introduction

Substantial prenatal mortality has been observed in all mammals studied, although there are significant differences between species in the extent and timing of the death. There are two reasons why it is important to define the causes of such loss. First, it is of fundamental interest to understand why prenatal loss continues to occur despite natural selection for efficient reproduction. Second, there may be practical applications arising from such knowledge if it can be used to increase the survival of embryos. Great distress is caused to those people who suffer infertility, particularly if it is a result of repeated miscarriage. Furthermore, a substantial economic loss follows prenatal death in farm animals. This death leads to a reduction in litter size in pigs and prolific sheep and, in cattle and sheep with only one ovulation at each oestrus, an increased interval between births.

The causes of prenatal mortality have been

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C. J. Ashworth, D. I. Sales and I. Wilmut

Summary. Plasma progesterone concentration and embryo survival were determined during successive pregnancies in ewes throughout one breeding season. The probability of an embryo surviving was associated with the progesterone concentration on the days around ovulation, with the timing of the increase from periovulatory to luteal values, and with the rate at which progesterone concentrations increased. Individual embryo survival decreased both as the number of corpora lutea increased, and towards the end of the breeding season; the latter effect could be explained entirely by differences in progesterone concentration. Considerable variation in progesterone secretion and in embryo survival was observed within the same ewes during successive pregnancies. Such variability in progesterone concentrations during early pregnancy may be a cause of some embryo mortality.

Keywords: embryo survival; fertility; progesterone; sheep; season

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I. Wilmut, M. L. Hooper and J. P. Simons

1 AFRC Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS, UK; and2Department of Pathology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK

Keywords: transgenic animals; gene targeting; reproduction

Contents

Introduction

Methods of genetic manipulation

Microinjection

Retroviral vectors

Embryonal stem cells

Spermatozoa as vectors

Factors affecting the expression of transgenes

Choice of species

Assays for transgene expression

Research strategies using genetically manipulated animals

Ablation of tissues

Cell lineages

Identification of new genes

Modification of gene function

Disease models

Other uses of oncogenes in transgenic animals

Somatic gene therapy

Research in reproduction

Embryogenesis

Homoeobox-containing genes

Genomic imprinting

Hypogonadal mice and the GnRH gene

Development of the reproductive tract

Lactation

Spermatogenesis

Conclusions

Introduction

During the past decade, revolutionary new opportunities in biological research have been created by bringing together techniques developed in two very different areas of biology: molecular biology and mammalian embryo manipulation. A gene from

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L. C. Smith, I. Wilmut and J. D. West

Summary. Karyoplasts derived from mouse embryos at the initial and final stages of the first or second mitotic interphase were fused to early and late enucleated 1-cell embryos. The time of cleavage of reconstituted and control embryos was recorded at 1-h or 8-h intervals after manipulation. This enabled assessment of nuclear and cytoplasmic control over the mitotic apparatus of the 1-cell embryo. Early nuclei from 1- or 2-cell embryos fused to late enucleated embryos delayed cleavage but for only a few hours. However, late nuclei fused to early enucleated embryos were unable to advance the cytoplasmic timing of the next cleavage division. Furthermore, these reconstituted embryos stayed in interphase longer than did controls and many embryos with nuclei derived from late 2-cell embryos failed to cleave. These findings suggest that, allowing for a short period, early nuclei can synchronize with late cytoplasm with no major damage to the cleavage apparatus. It is proposed that this period is required for the completion of DNA synthesis by the early nuclei. However, late nuclei cannot induce mitosis before the expected cytoplasmic time, and, with 2-cell karyoplasts, this interaction causes many embryos to 'block' in interphase, without cleaving, suggesting incompatible nucleo-cytoplasmic interactions between late 2-cell karyoplast and early 1-cell stage cytoplasm.

Keywords: embryo; nuclear transfer; cleavage; mouse

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L. C. Smith, I. Wilmut and R. H. F. Hunter

Summary. Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.

Keywords: embryo; nuclear transfer; cell cycle; mouse

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KH Campbell, P Loi, PJ Otaegui and I Wilmut

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.