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A H Handyside

Publications and source records attributed to A H Handyside.

89 records · Page 5Linked to original sources
PubMed1989

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro.

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Blastocyst↗
PubMed1988

Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere.

Single blastomeres were isolated from zona-free 8-cell mouse embryos and assayed for X-linked hypoxanthine phosphoribosyl transferase (HPRT) activity and autosome-linked adenine phosphoribosyl transferase (APRT) activity. At this stage of development both X chromosomes are active in female embryos. Hence, a bimodal distribution of HPRT: APRT ratios, corresponding to male (XY) and female (XX) biopsied samples, was observed due to the 2-fold difference in gene dosage for HPRT activity. Batches of putative male and female embryos identified in this way were transferred to pseudopregnant recipient females. Development of the seven-eighths embryos was equivalent to that of control zona-free intact embryos. Sex determination by measurement of X-linked gene dosage was accurate and rapid enough to allow transfer of embryos of known sex without the need for cryopreservation.

Adenine Phosphoribosyltransferase↗
PubMed1984

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.

Animals↗
PubMed1983

Reassortment of cells according to position in mouse morulae.

Sixteen-cell mouse morulae were disaggregated and blastomeres originally occupying outer or inner positions were separated. Outer, inner, or unsorted populations of blastomeres were labeled with either trinitrobenzene sulphonic acid (TNP) or fluorescein isothiocyanate (FITC) and individual blastomeres aggregated to unlabelled partially decompact eight- to ten-cell morulae. After up to 6 h in culture, the positions of the labelled blastomeres within the aggregates were examined. The combined results demonstrated that between 86 and 92% of outer cells remained on the surface of the aggregate and flattened into extensive polygonal shapes, whereas 76-77% of the inner cells had become engulfed by the host morula cells and retained their initial spherical shape. Using unsorted cells, 33-37% were internalised, which is compatible with the most recent estimates of the presence of six to eight inner cells at the 16-cell stage. The possibility that differential adhesiveness of the outer and inner cells is involved in the allocation of cells to the trophectoderm and inner cell mass of the blastocyst is discussed.

Animals↗
PubMed1983

Establishment of pluripotential cell lines from haploid mouse embryos.

Eggs from 129 SvE and (C57BL x CBA)F1 hybrid female mice were activated parthenogenetically following their exposure to a 7% solution of ethanol in PBS. Only the haploid class which developed a single pronucleus following second polar body extrusion was examined further. These eggs were transferred to suitable recipients and 'delayed' blastocysts subsequently recovered. The 'delayed' blastocysts were explanted into tissue culture and a total of four haploid-derived pluripotent cell lines established from individual embryos. Chromosome analysis of morulae revealed that over 80% contained only haploid mitoses. However, chromosome analysis of early passage cell lines revealed that all were diploid with a modal number of 40 chromosomes. When transplanted into syngeneic hosts, all lines formed well-differentiated teratocarcinomas. This technique provides a source of homozygous diploid cell lines of parthenogenetic origin.

Animals↗
PubMed1982

The developmental potential of mouse 16-cell blastomeres.

Based on the criteria of relative size and cell surface polarization, subpopulations of outer (larger, polar) and inner (smaller, apolar) blastomeres have been isolated from mouse 16-cell morulae and reaggregated in groups of 16 cells, and the developmental potential of the aggregates has been assessed both in vitro and in vivo. Aggregates of outer, inner, or mixed groups of cells all formed blastocysts which outgrew and contained both characteristic trophectoderm and alkaline phosphatase positive inner cell masses in vitro. However, significant differences were observed in the timing of blastocyst formation, depending on cell type. Aggregates of outer cells recompacted more slowly, commenced fluid accumulation earlier, and contained more cells at the blastocyst stage than did inner cell aggregates. Aggregates containing both outer and inner cells were intermediate. Postimplantation development of blastocysts derived from aggregates of outer or inner cells, after transfer to pseudopregnant recipients, was normal and comparable to zona-intact control embryos. This relationship between the expression of different morphological and behavioural properties by the precursor cells of the trophectoderm and inner cell mass and the existence of totipotent cells in both outer and inner subpopulations is discussed.

Animals↗
PubMed1982

Effect of microvilli on lateral diffusion measurements made by the fluorescence photobleaching recovery technique.

To consider the effect of surface microvilli on measurements of lateral diffusion by fluorescence photobleaching recovery, we have measured the diffusion of the lipid probe 3,3'-dihexadecyl indocarbocyanine iodide on the villated main body and unvillated budding polar body of unfertilized mouse eggs. On the main body we found D = (6.41 +/- 0.62) x 10(-9) cm(2)/s with (77.0 +/- 2.1)% recovery, and on the budding polar body we found D = (7.05 +/- 0.75) x 10(-9) cm(2)/s with (84.7 +/- 1.3)% recovery. We thus find only slight differences in diffusion in the two regions.

Animals↗
PubMed1981

Immunofluorescence techniques for determining the numbers of inner and outer blastomeres in mouse morulae.

Methods are described for distinguishing inner and outer blastomeres of compact 8- to 32-cell mouse morulae. The first involves the selective labelling by immunofluorescent reagents of the exposed surface of the compact morula, disaggregation of the morula into single blastomeres and separation of these blastomeres into partially labelled (presumptive outer) and unlabeled (presumptive inner) populations. The second involves labelling blastomeres after disaggregation and is based on the recent observation that the sera on an isolated outer blastomere which originally contributed to the exposed surface of the intact morula labels more intensely than the remaining (non-exposed) surface of the blastomere. Analysis of the labelling patterns obtained from individual disaggregated morulae indicated that inner blastomeres were absent from compact 8-cell morulae, but increased in number throughout the next cleavage division until at the 16-cell stage the mean number of these blastomeres varied from 4.6 to 6.6 (with a range of 1--8) depending on the technique used. At later stages, the numbers of inner blastomeres can probably be accounted for by division of the inner cells at the 16-cell stage.

Animals↗
PubMed1980

X-chromosome inactivation in extra-embryonic membranes of diploid parthenogenetic mouse embryos demonstrated by differential staining.

In somatic cells of female mammals one of the two X chromosomes is genetically inactive and heterochromatic, resulting in dosage compensation for X-linked genes. In marsupials the paternally derived X chromosome is preferentially inactivated. In eutherian mammals, although either X chromosome can be inactivated at random in somatic cells, preferential inactivation of the paternally derived X chromosome has been demonstrated cytologically in mouse and rat yolk sac and mouse chorion and biochemically in mouse yolk sac, chorionic ectoderm and trophoblast. In mouse yolk sac the non-random element has been shown both biochemically and cytologically to be confined to the endoderm layer in which there is almost total paternal X-chromosome activity in the separated yolk sac layers of diploid parthenogenetic mouse embryos in which both X chromosomes are maternally derived. Kaufman et al. have demonstrated X inactivation in somatic cells of diploid parthenogenetic embryos, and we have used a modification of Kanda's method, which renders the presumptive inactive X dark staining, to reveal an inactive X chromosome in both endoderm and mesoderm layers of separated yolk sacs from parthenogenones. Thus even in tissues in which there is normally total non-random paternal X inactivation, in the absence of a paternally derived X chromosome a maternally derived X can be inactivated.

Animals↗
PubMed1980

Distribution of antibody- and lectin-binding sites on dissociated blastomeres from mouse morulae: evidence for polarization at compaction.

The distribution of binding sites for rabbit anti-species antiserum, Concanavalin A (Con A) and peanut agglutinin (PNA) on dissociated blastomeres from 2- to 16-cell mouse embryos has been investigated using direct and indirect fluorescence techniques. With each ligand, paraformaldehyde-fixed blastomeres from 2- to 8-cell precompact embryos were uniformly surface labelled; the majority (77%) of late compact 8-cell blastomeres showed quantitative polarization of surface labelling; and 16-cell blastomeres were either polarized (53.3%) or uniformly surface labelled. Binding of fluorescein-conjugated PNA increased at the 16 cell stage. Labelling patterns on unfixed blastomeres were similar to those on fixed blastomeres except that surface label was patched and became internalized, most rapidly from the less heavily labelled areas of 8- and 16-cell blastomeres. Quantitative polarization of binding sites at postcompaction stages was detected after (i) fixation, (ii) pretreatment and labelling in the presence of azide, cytochalasin D and/or colcemid, or (iii) labelling with monovalent Fab1 antibody fragments. It is probably due, therefore, to the presence of microvilli at the heavily labelled pole, which increase surface area and are known to become to the outer surface of the compact morula (Ducibella, Ukena, Karnovsky & Anderson, 1977). The possibility that the cleavage of polarized blastomeres into dissimilar daughter blastomeres could provide a mechanism for the spatial differentiation of the inner cell mass and trophectoderm of the blastocyst is briefly discussed.

Animals↗
PubMed1979

The effect of prolonged decompaction on the development of the preimplantation mouse embryo.

A rabbit antiserum to a mouse embryonal carcinoma cell line blocks compaction of cleaving mouse embryos. Cell division is not affected up to the 32-cell stage but intracellular junctions fail to develop. Removal of the antibody at this stage permits compaction to occur and a normal blastocyst develops. Prolonged decompaction beyond the 32-cell embryo results in an increasing proportion of malformed blastocysts in which trophectodermal cells predominate and functional inner cell mass (ICM) cells are reduced or absent. The relationship of compaction to the generation of ICM and trophectoderm lineages in the intact embryo is discussed.

Animals↗
PubMed1978

Time of commitment of inside cells isolated from preimplantation mouse embryos.

Groups of inside cells (ICs) and inner cell masses (ICMs) were isolated from individual mouse embryos between the late morula and 3 1/2-day expanded blastocyst stages using a modified immunosurgical procedure, and their purity and developmental potential were assessed in vitro. Several different techniques failed to detect the presence of viable contaminating outside cells on ICs isolated from any of the stages studied. The numbers of inside cells isolated from the earlier stages, counted in air-dried preparations, were considerably higher than previous estimates from serial sections; whereas the numbers isolated from expanded blastocysts were in reasonable agreement. Thus the proportion of inside cells recovered by immunosurgery decreases over this period of development. In view of the evidence that inside cells divide at a faster rate than outside cells at these stages, it is argued that there may be an outward movement of inside cells capable of forming trophectoderm, during expansion of the blastocyst. ICs and ICMs in vitro were observed to develop in one of two distinct ways according to the stage at which they were isolated. ICs from late morulae and some early cavitating blastocysts formed blastocyst-like vesicles over a period of 24--36 h in culture. The presence of trophectoderm cells in these vesicles was confirmed by the persistence of giant cells after ectopic transfer. In contrast ICs from a minority of early cavitating blastocysts, and all ICMs from 3 1/2-day expanded blastocysts did not form vesicles, but proliferated endoderm-like cells. Thus at least some inside cells do not appear to lose the capacity to form trophectoderm and do not become committed to an ICM fate until after the initial formation of the blastocoel cavity.

Animals↗
PubMed1978

Temporal and spatial patterns of the synthesis of tissue-specific polypeptides in the preimplantation mouse embryo.

Mouse embryos at different morphological stages in the transition from morula to blastocyst were analysed for the qualitative pattern of their polypeptide synthesis. The first appearance of the individual species of polypeptides specific to trophectoderm or ICM occurred progressively over this developmental period. Populations of inside cells recovered from morulae and early blastocysts synthesized those polypeptides previously shown to be characteristic of ICMs at 31/2 days, but not those characteristic of trophectoderm.

Animals↗
PubMed1977

Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts.

Inner cell masses (ICMs) immunosurgically-isolated from 31/2-day mouse blastocysts were examined for trophoblast cell contamination and developmental capacity. Blastocysts were preincubated in rabbit anti-mouse antiserum, washed thoroughly and then incubated in complement. The ICMs were then easily dissected by drawing through a fine pipette. Various experiments confirmed that the trophectoderm had been completely removed by this treatment. Firstly, the ICMs did not bind a fluorescein-conjugated antibody directed against rabbit IgG, indicating the absence of cells exposed to the rabbit antiserum during the immunosurgical procedure. Secondly, ICMs dissected from blastocysts preincubated in a suspension of melanin granules did not include any of the trophoblast cells that had phagocytosed the granules. And, thirdly, the protein synthetic profile of these ICMs was similar to microsurgically dissected ICMs, and in particular, trophoblast specific spots were absent. The developmental capacity of immunosurgically-isolated ICMs was tested by injecting them into blastocysts and transferring to the uterus of 2 1/2-day pseudopregnant recipients. Extensive chimaerism was detected in the majority of implants, 5-6 days after transfer, but only in ICM-derived tissues. This demonstrates both the lack of trophoblast cell contamination and functional viability of these ICMs.

Animals↗
PubMedDate not supplied

Preimplantation genetic diagnosis of severe inherited skin diseases.

Considerable progress has been made recently in elucidating the molecular pathology underlying several forms of inherited skin diseases. One of the most immediate benefits of these discoveries has been the development of DNA-based prenatal diagnosis in pregnancies at risk for recurrence of a particular disorder. In less than 2 decades, prenatal testing has progressed from mid-trimester fetal skin biopsies or protein analysis in a limited number of conditions to first trimester chorionic villus sampling in a much broader range of genodermatoses. Advances in in vitro fertilization protocols and embryo manipulation technology have further led to the feasibility of even earlier prenatal diagnosis through preimplantation genetic diagnosis. This article details some of the recent advances in genetic skin disease research relevant to prenatal diagnosis and explores the possibilities and practicalities of preimplantation genetic diagnosis in the prevention of these conditions.

Animals↗