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R L Gardner

Publications and source records attributed to R L Gardner.

At least 55 records · Page 3Linked to original sources
PubMed1993

Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst.

Prominent among the various types of cell that differentiate from the trophectoderm of the mouse blastocyst are trophoblastic giant cells. Repeated endoreduplication of the genome accompanies the growth of these cells, which have been shown to be polytene rather than polyploid. Early stages in giant transformation have been examined, mainly in the mural trophectoderm of the implanting blastocyst which gives rise to the primary trophoblastic giant cells. One confusing issue is whether these early stages include the onset of endoreduplication of the genome. This issue has been addressed in the present study by comparing the DNA content of nuclei in isolated trophectoderm and ICM tissue rather than, as previously, by relating measurements on air-dry preparations of entire blastocysts to those of adult liver. The results, particularly those from delayed and reactivated blastocysts, show that genome endoreduplication is not an obligatory early event in the transformation of mural trophectoderm cells.

Animals↗
PubMed1993

Extrinsic factors in cellular differentiation.

An impressive feature of cellular differentiation in metazoa is its stability. This has led to widespread acceptance of the view that the determined state is a heritable property of individual cells. There are, in fact, rather few types of cell for which this has been demonstrated convincingly. Not only is it clear that gene expression is subject to continuous regulation but there is also evidence for an increasing variety of cells that changes in differentiation can be induced by manipulating their environment. Such findings suggest that extrinsic factors may play a more significant role in maintaining the differentiated state of cells than is generally assumed.

Animals↗
PubMed1992

Is the anterior-posterior axis of the fetus specified before implantation in the mouse?

The mouse conceptus is generally held to be radially symmetrical about its embryonic-abembryonic axis from the blastocyst stage until the primitive streak appears at the beginning of gastrulation. However, this notion has been challenged by recent observations on conceptuses sectioned in utero which suggest that the blastocyst is already bilaterally symmetrical when it begins to implant. Accordingly, the blastocyst has been assigned an anterior-posterior axis which appears to persist through gastrulation and is claimed to coincide with the anterior-posterior axis of the future fetus in both orientation and polarity. In the present investigation the relationship between these two axes was examined in conceptuses dissected from the uterus early in gastrulation so that it could be determined more accurately than is possible in situ. The anterior-posterior axis of the conceptus and nascent fetus were found to be either parallel or antiparallel to each other, suggesting that while the orientation of the fetal axis may be specified at the blastocyst stage its polarity is not.

Animals↗
PubMed1992

Effects of diapause on lethal yellow (Ay/Ay) mouse embryos.

One of the problems in studying early acting recessive lethal genes is recognizing the homozygotes prior to their demise. Molecular probes can assist in this task, but their use generally requires removal of cells and consequent damage to the embryo, which may compromise its subsequent development. The present study was undertaken in an attempt to distinguish intact, living, lethal yellow (Ay/Ay) embryos from Ay/ae and ae/ae littermates before implantation by placing them in implantation delay or diapause. After 2 days in the reproductive tract of prepubertal females, the great majority of presumptive lethal Ay/Ay embryos has failed to hatch from the zona pellucida and they exhibited a marked deficiency of cells relative to controls, particularly in the inner cell mass. This argues against a stage-specific role for the gene in implantation.

Animals↗
PubMed1991

An investigation of the fate of cells transplanted orthotopically between morulae/nascent blastocysts in the mouse.

A technique has been devised for selectively replacing some inside cells (ICs) or outside cells (OCs) of late morulae/nascent blastocysts with corresponding cells from genetically dissimilar, synchronous embryos. The main purpose was to determine whether the inner cell mass (ICM) contributes cells to the overlying polar trophectoderm at any stage during the blastocyst phase of development. Notwithstanding the high incidence and level of chimaerism in ICM derivatives of post-implantation conceptuses obtained in the IC transplantation experiments, trophoblast tissue was composed entirely of host cells in the majority of cases. Even where a donor IC contribution to trophoblast was detected there were strong grounds for suspecting that it was due to tissue contamination rather than genuine chimaerism. Thus, not only did such contributions differ in both level and distribution from those produced by transplantation of OCs but they also varied markedly in frequency according to the day of gestation on which conceptuses were dissected. The possibility that ICs regularly colonize the polar trophectoderm but fail to persist there was excluded by the results of short-term transplantation experiments using an in-situ genetic marker. These findings offer no support for the hypothesis that the ICM serves as a population of stem cells for the trophectoderm as well as the primitive endoderm and ectoderm during normal development. The frequency of chimaerism was lower in OC than IC transplantation experiments. Nevertheless, in a substantial proportion of chimaeras, OCs colonized derivatives of the ICM. This is consistent with evidence from other studies that some outside cells divide differentially at 5th cleavage to produce an OC plus an IC rather than two OCs.

Animals↗
PubMed1991

Human trophectoderm biopsy and secretion of chorionic gonadotrophin.

We have previously developed a technique of trophectoderm biopsy to obtain cells from human blastocysts for preimplantation genetic diagnosis. To determine whether this technique affects the subsequent development of the blastocyst, 45 manipulated blastocysts were observed from days 3 to 14 in culture, the amount of human chorionic gonadotrophin (HCG) secreted by each embryo was measured and these results were compared with those of 26 non-manipulated controls documented in a previous study. A slit was made in the zona pellucida opposite the inner cell mass in 18 of the 45 blastocysts. This increased the rate of hatching but the other morphological changes up to day 14 were similar to those seen in the non-manipulated controls. There was no difference in the mean cumulative HCG secretion by these zona-slit controls (149.8 +/- 45.7 mIU/ml) compared to the non-manipulated controls (146.2 +/- 23.7 mIU/ml). A slit was also made in the zona pellicida of the other 27 blastocysts. Approximately 12-18 h later, in 18 blastocysts a biopsy of the herniating trophectoderm cells (5-30) was performed. The rate of hatching and adherence to the culture dish was similar to the non-manipulated controls. The mean cumulative HCG secretion decreased significantly (57.5 +/- 16.2 mIU/ml, P less than 0.01) after the biopsy procedure. However, if a small biopsy was performed (less than 10 cells removed) the decrease in HCG secretion (87.6 +/- 24.8 mIU/ml) was not significant, whereas when a large biopsy was performed (greater than 10 cells), HCG levels fell to 19.9 +/- 9.1 mIU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy↗
PubMed1991

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro.

Micromanipulation of human oocytes and embryos has provided new opportunities for both the treatment of infertility and the preimplantation diagnosis of genetic disease. It is important to determine whether manipulated embryos develop normally in vitro, as an indication of their suitability for transfer. However, at present there is little information on the development of non-manipulated embryos in vitro for comparison. We have therefore monitored morphological changes and human chorionic gonadotrophin (HCG) secretion in 36 non-manipulated human embryos, including 26 blastocysts and 10 cavitating morulae, daily from day 3 to day 14 of culture. Hatching was observed in 10 (38.5%) blastocysts and five of these adhered to the culture dish and appeared viable until day 14. The secretion of HCG was first detected on day 8, peaked at day 10 (51.11 +/- 8.7 mIU/ml) and then declined but was still detectable in four blastocysts on day 14. There was no overall difference in HCG secretion by hatched blastocysts and those which remained within the zona. However, those hatched blastocysts which showed adherence had significantly increased (P less than 0.05) HCG secretion. For individual blastocysts, the pattern of HCG secretion correlated well with the assessment of morphology. These data provide the basis for comparative studies of morphological changes and HCG secretion in manipulated embryos.

Blastocyst↗
PubMed1990

Trophectoderm biopsy in human blastocysts.

Trophectoderm biopsy was carried out on 47 human blastocysts. A slit was made in the zona pellucida opposite the inner cell mass by micromanipulative techniques. The human blastocyst zona offered more resistance to slitting compared to that of the mouse. After 18-24 h, controlled herniation of the trophectoderm cells was observed. These cells were biopsied when the diameter of the herniation was approximately equal to that of the blastocyst. The size of the slit and the stage of embryonic development at which slitting was performed were important for successful herniation to occur. After slitting, 76% of day 5-6 blastocysts showed herniation whilst only 42% of day 7-8 blastocysts herniated. Further development of the manipulated embryos was not apparently impaired, as hatching occurred in 44% of the former and 20% of the latter, compared with 18.1% in non-manipulated controls. The biopsied cells (approximately 10-30) usually remained in a clump but 14% formed vesicles on the day after biopsy. There was, however, no evidence of adherence to the dish or formation of monolayers. These results demonstrate the feasibility of trophectoderm biopsy in human blastocysts and that sufficient extra-embryonic material can be obtained by this technique for preimplantation diagnosis of genetic disorders.

Biopsy↗
PubMed1989

Effect of damage to the zona pellucida on development of preimplantation embryos in the mouse.

The effect on development of early mouse embryos of making a hair-line slit in the zona pellucida of approximately one-third its diameter was investigated. The rate of development to mid-gestation of operated zygotes and 2-cell embryos transferred directly to the oviduct was significantly lower than that of sham-operated or unoperated controls. However, the operation had no discernible effect on the development of 2-cell embryos that were cultured for 2 days prior to transfer to the uterus, or on embryos composed of 8 or more cells transferred directly to the oviduct. Zona slit zygotes and 2-cell embryos exhibited a significantly higher rate of anomalous development to the morula or blastocyst stage than controls following short-term transfer to the adult or immature oviduct. Such anomalies could not be attributed to damage of the embryos by leucocytes or bacteria entering through the wound in the zona. Rather, the typically non-spherical shape of slit zonae, together with the fact that some were empty on recovery, was consistent with operated embryos having been damaged by compression during passage through the oviduct. This suggests that, providing it is intact, the zona pellucida protects the early embryo from contraction of the oviductal musculature which is sufficient to lyse, arrest or extrude blastomeres prior to the formation of intercellular junctions. Hence, in experimental manipulations entailing damage to the zonae of early embryos, there may be a case for allowing them to form morulae in vitro prior to transfer, rather than returning them directly to the oviduct.

Animals↗
PubMed1989

Cell allocation and lineage in the early mouse embryo.

In the early mammalian embryo, initially asymmetric cell contacts appear to induce blastomere polarization, elements of which can persists through cytokinesis. This leads to the generation of inner and outer populations of blastomeres which may subsequently diverge as a result of residing in distinct microenvironments. Similar processes may account for the generation of primitive endoderm versus primitive ectoderm, and that of trophectoderm versus inner cell mass. However, if this is the case, the response of cells to positional cues must change as a function of either their previous positional history or the number of cycles they have completed. Once these primary tissues have been established, specific interactions between them lead to further cellular diversification.

Animals↗
PubMed1988

Multi-lineage 'stem' cells in the mammalian embryo.

The term 'stem' cell has acquired a rather more restricted meaning in cell biology than in embryology as a result of studies on the growth kinetics of renewing tissues in mature organisms. It is normally used in an embryological context as a synonym for 'progenitor' cell. Methods of establishing the existence of multi-lineage progenitor cells in mammals are examined briefly before the occurrence and properties of such cells in both embryonic and extra-embryonic tissues of the mouse conceptus are reviewed. Various attributes of 'stem' cells that can be obtained from outgrowths of blastocysts in vitro are also discussed.

Animals↗
PubMed1985

Clonal analysis of early mammalian development.

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.

Amnion↗
PubMed1985

Regeneration of endoderm from primitive ectoderm in the mouse embryo: fact or artifact?

The capacity of immunosurgically (IS) treated inner cell masses (ICMs) versus microsurgically (MS) isolated primitive ectoderms from blastocysts recovered on the 5th day of gestation to regenerate an external layer of endoderm cells in vitro was investigated. While the majority of IS-treated ICMs regenerated such a layer, MS-isolated ectoderms seldom did so. Examination of the two types of tissue fragments revealed that IS-treated ICMs almost invariably retained viable endoderm cells whereas MS-isolated ectoderms did so only exceptionally. The endoderm was found to be more than one cell layer thick in ICMs from 5th day blastocysts, suggesting that some endoderm cells survive IS because they are protected from exposure to antiserum. Typing of the endoderm layer that regenerated following IS treatment of recombinant ICMs composed of genetically dissimilar endoderm and ectoderm provided direct evidence that it originated from residual endoderm cells rather than the underlying ectoderm. Finally, blastocyst injection experiments confirmed that IS-treated ICMs behave like a mixture of ectoderm and endoderm tissue in vivo, and provided no support for the view that cells of the original and regenerated endoderm differ in developmental potential. These findings challenge earlier conclusions concerning cell lineage and determination in the primitive ectoderm that were based on development in vitro of IS-treated ICMs from giant blastocysts.

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