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J D West

Publications and source records attributed to J D West.

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

Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigenin-labelled probes.

DNA-DNA in situ hybridization, with two digoxigenin-labelled, chromosome-specific DNA probes, was used to determine the number of copies of a given chromosome in interphase nuclei and so identify putatively polyploid nuclei in histological sections of several mouse tissues. One hybridization site per diploid genome was expected for tissues with hemizygous markers: male mice hybridized with a Y chromosome probe (pY353/B) or hemizygous transgenic mice hybridized with a beta-globin probe (pM beta delta 2). Nuclei with more than one hybridization site were considered putative polyploids. Three groups of experiments were undertaken: (1) evaluation of the method, using mouse liver sections; (2) studies of tissues already known to contain polyploid nuclei, and (3) studies that resulted in the discovery that the mouse ovary contains polyploid nuclei. First, control studies showed that the ability to detect the target DNA sequences was affected by section thickness. Studies of nuclear ploidy in the developing mouse liver revealed a pattern similar to that established by previous studies using DNA content as a criterion for ploidy. At birth, only about 5% of the liver nuclei were polyploid; this increased to 10-15% by 10-20 days and was followed by a sharp increase in the frequency of tetraploid nuclei between 20 and 40 days (to about 35%) and a more gradual increase in higher order polyploid nuclei. Secondly, this technique was used to confirm that polyploid (mostly tetraploid) nuclei were present in the bladder epithelium, heart, uterine decidua and placental trophoblast. Higher order polyploidy was seen in large bone marrow cells (megakaryocytes) but not in the even larger trophoblast giant cells of the placenta, thus confirming previous claims that these cells are polytene rather than polyploid. Thirdly, putatively tetraploid nuclei were found in the ovarian follicle and corpus luteum. As far as we are aware, this is the first time polyploid nuclei have been reported for the mouse ovary.

Animals↗
PubMed1993

Inheritance of a meiotic abnormality that causes the ovulation of primary oocytes and the production of digynic triploid mice.

Previous studies have demonstrated that the LT/SvKau strain of mice ovulates a high proportion of oocytes as diploid primary oocytes rather than secondary oocytes. These ovulated primary oocytes are arrested at meiotic metaphase I but may be fertilized to produce digynic triploid embryos. In the present study, 40.4% of eggs analysed from LT/SvKau females were ovulated as primary oocytes, compared to 1.2% from control C57BL/Ws strain mothers. These two inbred strains were intercrossed to produce eight sets of F1, F2 and backcross females and the frequency of triploidy was investigated. The results are compatible with segregation of a co-dominant, autosomal gene that has a major influence on the incidence of triploidy. We suggest that the provisional gene symbol Poo (primary oocyte ovulation) be assigned to this gene, with alleles Poo(l) (the 'mutant' allele present in the LT/SvKau strain) and Poo(b) (the normal allele present in C57BL/Ws mice). Poo is incompletely penetrant and has variable expressivity because the proportion of oocytes ovulated as primary oocytes by LT/SvKau mice was variable and, in some cases, nil. In putative Poo(l)/Poo(b) heterozygotes the frequency of ovulated primary oocytes was increased only marginally (from 1.2% to 6.6%) by the presence of one copy of the Poo(l) allele, but this increase was found consistently (in two reciprocal F1 crosses) and was statistically significant. No evidence was found for tight genetic linkage between Poo and two Mendelian loci (brown on chromosome 4 and glucose phosphate isomerase on chromosome 7), that were segregating in the crosses. The Poo(l) mutant in the LT/SvKau strain of mice provides a valuable resource to study the cell and molecular biology of mammalian oocyte maturation and the control of meiosis.

Alleles↗
PubMed1993

A genetically defined animal model of anembryonic pregnancy.

Mutant mouse conceptuses, homozygous for a defect in the gene encoding the glycolytic enzyme, glucose phosphate isomerase (GPI) died in utero by 9 1/2 days post coitum. By this stage there was no normal embryo, but trophoblast and some extra-embryonic membranes usually survived. The morphology of these genetically determined, anembryonic conceptuses was similar to some of the sporadic cases of anembryonic conceptuses that occurred in control crosses. These similarities suggest that an understanding of the cause of death of the homozygous mutant embryos might shed light on the aetiology of some of the cases of sporadic anembryonic conceptuses in both mouse and man. At implantation, the conceptus depends on anaerobic glycolysis for its energy production and any shortage of substrates would compromise development. It is argued that, in the absence of efficient glycolysis, a homozygous null embryo would be unable to produce sufficient energy to develop normally beyond the egg cylinder stage. However, the outer part of the conceptus might survive if nutrients and oxygen were available from the surrounding maternal tissue to produce energy by the aerobic tricarboxylic acid cycle. The homozygous GPI-null conceptuses may provide a useful animal model for some types of human anembryonic pregnancy.

Animals↗
PubMed1993

Fetal growth, gestation length and phosphoglucomutase-1 phenotype.

This study investigates reports that phosphoglucomutase-1 (PGM1) phenotype is associated with fetal growth and gestation length. A total of 350 women were studied, 234 having uncomplicated pregnancies and 114 with a baby weighing greater than 90th centile, corrected for parity, gestation and fetal sex. All women had gestation confirmed by early ultrasound. Conventional cellulose acetate electrophoresis was used to distinguish the three common PGM1 phenotypes and polyacrylamide gel isoelectric focusing to distinguish the ten PGM1 subtypes. Neither PGM1 phenotype nor subtype were found to be associated with gestation length or standardised birth weight. Logistic regression, where maternal age, parity, fetal sex, maternal weight, gestation and smoking were introduced as explanatory variables in addition to PGM1 phenotype testing against the dependent variables birth weight, standardised birth weight and gestation length, did not show differences related to PGM1 phenotype. Two possible reasons for the discrepancy with previously published data are discussed. We conclude that the study provides no support for the belief that PGM1 phenotype is related to fetal growth or gestation length and that the original observations could have arisen as a result of statistical artefact due to multiple testing.

Biomarkers↗
PubMed1992

Dominant hemimelia and En-1 on mouse chromosome 1 are not allelic.

Previous studies have shown that En-1, a homeobox-containing gene, maps close to or at the Dh locus in the mouse. Since homeobox-containing genes are key genes in the control of development the close proximity of En-1 to the developmentally significant gene Dh raised the possibility that the Dh mutation represented a mutant allele of En-1. A genetic analysis involving En-1, Dh, and other chromosome 1 markers (Emv-17, ln and Pep-3) shows that although Dh and En-1 are closely linked they are separable by recombination (4/563). The likely gene order and recombination frequencies of these loci are: ln (5.2 +/- 0.9) Emv-17 (1.1 +/- 0.4) Dh (0.7 +/- 0.4) En-1 (3.0 +/- 0.7) Pep-3. This shows that Dh is not a mutant allele of En-1.

Animals↗
PubMed1992

Electrofusion of mouse embryos results in uniform tetraploidy and not tetraploid/diploid mosaicism.

Some previous attempts to produce tetraploids experimentally have resulted in a proportion of treated embryos becoming 2n/4n mosaics at a frequency which may be as high as 20%, when using cytochalasin B as a fusigenic stimulus and cytogenetic techniques to identify putative tetraploid embryos. To investigate the possible occurrence of 4n/2n mosaicism, tetraploid embryos were produced by electrofusion, a process which allows adjacent blastomeres at the 2-cell stage to fuse following exposure to electric field pulses. Embryos used for electrofusion were hemizygous for a transgene consisting of approximately 1000 copies of the mouse beta-globin gene. After in situ hybridization, one hybridization signal is expected per diploid genome. Tetraploid cells in 7.5-, 8.5-, 9.5- and 10.5-day-old conceptuses were distinguished from diploid cells by performing in situ hybridization on histological sections. The frequency of nuclei with two hybridization signals in the 'hemizygous' tetraploid embryos was compared to diploid embryos which were either hemizygous or homozygous for the beta-globin transgene. Comparison of the frequency of nuclei with two hybridization signals between tissues of 'hemizygous' tetraploid conceptuses and homozygous diploid conceptuses showed no significant difference, which implies that the tissues in the tetraploid conceptuses were uniformly tetraploid. No evidence was found to suggest that electrofusion results in 2n/4n mosaicism.

Animals↗
PubMed1992

The prion protein gene: a role in mouse embryogenesis?

The neural membrane glycoprotein PrP (prion protein) has a key role in the development of scrapie and related neurodegenerative diseases. During pathogenesis, PrP accumulates in and around cells of the brain from which it can be isolated in a disease-specific, protease-resistant form. Although the involvement of PrP in the pathology of these diseases has long been known, the normal function of PrP remains unknown. Previous studies have shown that the PrP gene is expressed tissue specifically in adult animals, the highest levels in the brain, with intermediate levels in heart and lung and low levels in spleen. Prenatally, PrP mRNA has been detected in the brain of rat and hamster just prior to birth. In this study we have examined the expression of the PrP gene during mouse embryonic development by in situ hybridisation and observed dramatic regional and temporal gene expression in the embryo. Transcripts were detected in developing brain and spinal cord by 13.5 days. In addition, PrP gene expression was detected in the peripheral nervous system, in ganglia and nerve trunks of the sympathetic nervous system and neural cell populations of sensory organs. Expression of the PrP gene was not limited to neuronal cells, but was also detected in specific non-neuronal cell populations of the 13.5 and 16.5 day embryos and in extra-embryonic tissues from 6.5 days. This cell-specific expression suggests a pleiotropic role for PrP during development.

Animals↗
PubMed1991

Expression of glucose phosphate isomerase in interspecific hybrid (Mus musculus x Mus caroli) mouse embryos.

Hybrid Mus musculus x Mus caroli embryos were produced by inseminating M. musculus (C57BL/OlaWs) females with M. caroli sperm. Control M. caroli embryos developed more rapidly than did control M. musculus embryos and implanted approximately 1 day earlier. At 1 1/2 days, both the hybrid embryos and those of the maternal species (M. musculus) had cleaved to the 2-cell stage. By 2 1/2 days some of the hybrids were retarded compared to M. musculus, and by 3 1/2 days most were lagging behind. This is consistent with the idea that the rate of development of hybrid embryos declines once it becomes dependent on embryo-coded gene products. We have used this difference in rate of preimplantation development, between hybrid and M. musculus embryos, to try to determine whether the activation of embryonic Gpi-1s genes, that encode glucose phosphate isomerase (GPI-1), is age-related or stage-related. In control M. musculus embryos (both mated and Al groups), the GPI-1AB and GPI-1A allozyme, indicative of paternal gene expression, were detected in 7 of 9 samples of 3 1/2-day compacted morula stage embryos and were seen in all 19 samples of 3 1/2-day blastocysts. In hybrid embryos, these allozymes were detected 1 day later. They were not detected in any 3 1/2-day samples (12 samples of compacted morulae) but were consistently detected at 4 1/2 days (4 samples of blastocysts and 2 samples of uncompacted morulae). Our interpretation of the results is that gene activation in hybrid embryos is stage-specific, rather than age-specific, and probably begins around the 8-cell stage, with detectable levels of enzyme accumulating later. Analysis of GPI-1 electrophoresis indicated that both the paternal (M. caroli) and maternal (M. musculus) Gpi-1s alleles were equally expressed in hybrid embryos and that the paternally derived allele was not activated before the maternally derived allele.

Animals↗
PubMed1990

Relevance to prenatal diagnosis of the identification of a human Y/autosome translocation by Y-chromosome-specific in situ hybridisation.

Routine cytogenetic analysis of an amniotic fluid sample revealed a large brightly fluorescent region in the short arm of chromosome 14 in an otherwise normal male karyotype (46,XY,14p+ + +). This site was also present in the paternal karyotype. In situ hybridisation to a Y-chromosome-specific DNA probe confirmed that the father had a Y/14 translocation. The incidence of two hybridisation bodies (large hybridisation sites), detecting both the translocated Y chromatin and the normal Y chromosome, was lower in interphase nuclei (44.3%) than in metaphase spreads (95.2%). The relevance of these observations to the potential use of in situ hybridisation to interphase nuclei for prenatal diagnosis is discussed.

Adult↗
PubMed1990

Control of first cleavage in single-cell reconstituted mouse embryos.

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.

Animals↗
PubMed1989

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei.

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.

Adult↗
PubMed1989

The use of DNA probes in preimplantation and prenatal diagnosis.

DNA probes are now widely used for prenatal diagnosis, but the prospect of preimplantation diagnosis of genetic disorders requires the development of sensitive genetic tests that can be performed on small numbers of cells removed from a preimplantation-stage pre-embryo. The sensitivity of molecular tests can now be increased by specifically amplifying the target DNA with the polymerase chain reaction. In situ hybridisation with chromosome-specific DNA probes to repeated sequences also permits the detection of particular numerical chromosome aberrations or the distinction of male and female pre-embryos when only a few interphase nuclei are available. We have used in situ hybridisation to a Y chromosome-specific DNA probe to sex preimplantation-stage pre-embryos and to sex fetuses from samples of chorionic villus cells, amniotic fluid cells, and fetal blood. These two approaches (amplification of target DNA and in situ hybridisation) provide suitable tests for improving prenatal diagnosis particularly when few cells are available and they offer the possibility of tests suitable for preimplantation diagnosis.

Blastocyst↗
PubMed1989

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation.

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.

Autoradiography↗
PubMed1989

Non-additive inheritance of glucose phosphate isomerase activity in mice heterozygous at the Gpi-1s structural locus.

The activity of blood glucose phosphate isomerase (GPI-1) in mice heterozygous for various alleles at the Gpi-1s structural locus (heterozygotes a/b, a/c and b/c) was significantly higher than expected, on the basis of additive inheritance, from the levels in parental homozygotes. Moreover, the GPI-1 activity was higher in a/b heterozygotes than in either parent (heterosis). Studies of heat stability with kidney homogenates revealed that the relative stabilities of GPI-1 dimers was AA greater than AB greater than BB greater than AC greater than or equal to BC greater than CC. Differences in dimer stabilities in vivo would affect the total GPI-1 levels in heterozygotes and could account for non-additive inheritance but would be insufficient to explain heterosis for GPI-1 activity. Other possible contributing factors include unequal production or stability of monomers, or higher catalytic activity of heterodimers. Monomers could also associate non-randomly but this would not be sufficient to explain heterosis. It is clear that non-additive inheritance patterns may be produced by variants of either structural or regulatory genes.

Animals↗
PubMed1989

Glucose phosphate isomerase activity in mouse and human eggs and pre-embryos.

We have measured the activity of glucose phosphate isomerase (GPI-1) in 12 unfertilized human eggs and five human pre-embryos relative to the GPI-1 activity in C57BL/OlaWs mouse eggs. The GPI-1 activity in the human eggs was approximately 6 times that in the C57BL mouse eggs. This implies that human eggs have approximately twice the activity per unit volume of the C57BL mouse eggs but no more than certain other strains of mice. The activity in five human pre-embryos, the most advanced of which was an early blastocyst, was similar to that seen in the human eggs. No change in GPI-1 activity was seen in mouse pre-embryos up to 2 1/2 days (8- to 12-cell stage) but the activity had declined by 3 1/2 days (compacted morula and early blastocyst stages). It seems that high levels of GPI-1 activity are maintained during the early preimplantation development of both species.

Animals↗
PubMed1989

Genetic differences in glucose phosphate isomerase activity among mouse embryos.

We have compared mouse embryos of three heterozygous, congenic genotypes (with high, medium and low levels of oocyte-coded glucose phosphate isomerase (GPI-1) activity respectively) to test whether 1) the survival time of oocyte-coded GPI-1 activity in the early embryo is affected by its activity level in the oocyte and 2) whether embryo-coded GPI-1 is detected earlier in embryos that inherit low levels of oocyte-coded GPI-1. The oocyte-coded GPI-1 was entirely GPI-1A allozyme in the high and medium groups but was the less stable GPI-1C allozyme in the low group. We determined total GPI-1 activity and the ratio of different GPI-1 allozymes in early embryos and calculated the activity of oocyte-coded and embryo-coded GPI-1. In all three groups, the oocyte-coded enzyme activity remained at a more or less constant level for the first 21 1/2 days. Some oocyte-coded GPI-1 remained in 4 1/2 day embryos from the high and medium groups but was gone by 5 1/2 days. Very little remained in 4 1/2 day embryos that inherited low levels of a less stable form of the enzyme (GPI-1C allozyme). Despite a 4- to 5-fold difference in initial oocyte-coded GPI-1 activity, no differences were seen among the three genotypically distinct groups of embryos in the time of activation of the embryonic Gpi-1s genes. The embryo-coded GPI-1 was first detectable in 3 1/2 day compacted morulae in all three groups. The level of oocyte-coded GPI-1, in the high group, when embryo-coded GPI-1 was first detected was higher than the level in the low group at any stage prior to detection of embryo-coded GPI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Sexing whole human pre-embryos by in-situ hybridization with a Y-chromosome specific DNA probe.

We have used DNA-DNA in-situ hybridization with a DNA probe for the human Y-chromosome to distinguish between male and female human pre-embryos. Both biotinylated and tritiated Y-probes worked well on control cell cultures where 100 interphase nuclei were scored. Morphologically normal pre-embryos could be sexed with confidence with the tritiated Y-probe but the biotin results were less reliable (although only a few pre-embryos were analysed in this way). Early cleavage stage pre-embryos had large nuclei with relatively diffuse Y-bodies and were more difficult to score with the biotinylated Y-probe. Morphologically abnormal pre-embryos often had large nuclei with multiple Y-bodies (presumably polyploid nuclei) or small nuclei with no Y-bodies (possibly fragmenting nuclei). In all, 38 cleaving and two non-cleaving pre-embryos were analysed. The incidence of false positive and false negative cells seen after hybridization of tritiated Y-probes to control lymphocyte cultures suggests that it should normally be possible to distinguish morphologically normal male and female pre-embryos with samples of three to six interphase nuclei.

Blastocyst↗