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Biomedical subjects

J C Harper

Publications and source records attributed to J C Harper.

50 records · Page 3Linked to original sources
PubMed1995

Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos.

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6-10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analyzed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.

Blastomeres↗
PubMed1995

The use of fluorescent in-situ hybridization (FISH) for the analysis of in-vitro fertilization embryos: a diagnostic tool for the infertile couple.

We use triple colour fluorescent in-situ hybridization (FISH) to sex human embryos for preimplantation diagnosis of X-linked disease, to analyse chromosome numbers in embryos donated for research purposes and as a diagnostic tool for patients undergoing infertility treatment, especially in cases where abnormal embryo development occurs. We have reported on the use of FISH in a case where all embryos showed accelerated cleavage. Here we report on the use of triple colour FISH in a case where five out of seven oocytes were multi-nucleated when examined for pronuclei. The embryos were spread whole using HCl/Tween 20 and triple colour FISH performed with probes for chromosomes X, Y and 1 in a 2 h procedure. Two embryos were normal for the probes used, and three showed abnormalities, including one 4-cell embryo where all nuclei were X,X,X,Y,1,1,1,1. FISH indicated that fertilization had occurred, but that the majority of embryos were abnormal confirming that such embryos should not be considered for transfer. In these cases, or where there is recurrent in-vitro fertilization failure or spontaneous abortions, embryos in future cycles can be examined using FISH to ascertain the level of chromosome abnormality which may aid future infertility treatment.

Blastocyst↗
PubMed1994

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation.

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.

Blastocyst↗
PubMed1994

Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization.

PURPOSE: Our purpose was to assess the clinical application of dual fluorescent in situ hybridization (FISH) for the diagnosis of sex in the human preimplantation embryo. RESULTS: Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male:female, 60:40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer. CONCLUSION: Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.

Adult↗
PubMed1994

Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes.

Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos for preimplantation diagnosis of X-linked disease. With a modified spreading method and directly labelled fluorescent DNA probes, we have examined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggregated and 98 intact blastomeres obtained. The spreading efficiency was 96% and FISH signals were obtained from 97% of nuclei. In all cases, sibling blastomeres from the same embryo were the same sex. Mosaicism was observed in some embryos. Five cells which lysed during the disaggregation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor and multiple signals were observed so that reliable diagnosis of sex was not possible. The data reported here confirm that by using an improved spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sexing embryos for preimplantation diagnosis of X-linked disease.

Blastomeres↗
PubMed1994

Detection of fertilization in embryos with accelerated cleavage by fluorescent in-situ hybridization (FISH).

Embryos from a couple undergoing routine in-vitro fertilization for unexplained infertility had shown cleavage by day 1 in two consecutive cycles. The response of the woman to fertility drugs had been normal, and there were no known sperm abnormalities. In a subsequent cycle, accelerated cleavage occurred again and we used a modified method of spreading whole embryos and dual fluorescent in-situ hybridization (FISH) with directly labelled probes for chromosomes X and Y to determine if fertilization had occurred in these embryos. Nine oocytes were collected, five of which had cleaved when examined for pronuclei early on day 1 following late insemination. Pronuclei were observed in one of the remaining oocytes, but in this case, three were present. On day 2, all of the oocytes/embryos had cleaved and two were transferred to the patient. The remaining seven were spread for FISH analysis but nuclei were only obtained from five. In three, a Y signal was detected, indicating that fertilization had occurred. In all five embryos, a wide range of X chromosome signals were observed. These data suggest that the embryos had undergone abnormal fertilization and accelerated cleavage.

Adult↗
PubMed1991

Characteristics of catecholamine release from adrenal chromaffin cells cultured in medium containing ethanol--I. Spontaneous and K(+)-induced release.

Bovine adrenal chromaffin cells were dissociated and grown in primary culture for 9 days. Three days after plating, half the cultures were grown in a medium containing 200 mM ethanol and the other half in a control medium, for a further 6 days. The catecholamine content of the ethanol-treated cells was increased after 6 days of ethanol treatment, compared to control cells and there was a slight reduction in protein and DNA content. An enhanced spontaneous release of catecholamines was seen, which was Ca(2+)-dependent and was inhibited by cadmium but not by the organic dihydropyridine Ca2+ antagonist nitrendipine. The fraction of catecholamines released by K+ was also enhanced in ethanol-treated preparations, particularly at high K+ or Ca2+ concentrations. Release induced by K+ was sensitive to inhibition by both cadmium and organic dihydropyridine Ca2+ antagonists. The results show many similarities with changes observed in catecholaminergic transmission in rat brain during the development of ethanol physical dependence.

Adrenal Glands↗
PubMed1991

Characteristics of catecholamine release from adrenal chromaffin cells cultured in medium containing ethanol--II. Carbachol and veratrine-induced release.

Dissociated bovine adrenal chromaffin cells were grown in culture either in control medium or in medium containing ethanol (200 mM) and the release of catecholamines induced by veratrine and carbachol was then studied. Cells grown in ethanol showed greater spontaneous release of catecholamines, but both carbachol and veratrine evoked release of a smaller maximum fraction of stored catecholamines than that from control cells. Spontaneous catecholamine release as well as that induced by veratrine were sensitive to inhibition by tetrodotoxin. Catecholamine release induced by veratrine could be prevented almost completely by the Ca2+ channel blocker cadmium, but not to any great extent by the organic dihydropyridine Ca2+ antagonist nitrendipine. Carbachol-induced catecholamine release was similarly resistant to inhibition by nitrendipine, but was inhibited by cadmium. The results suggest that cell cultures grown in ethanol-containing medium show many alterations in the characteristics of catecholamine release. Alterations in receptor- and voltage-operated ion channels in the membrane of ethanol-treated cells may underlie these changes.

Adrenal Glands↗
PubMed1990

Development of tolerance to ethanol in cultured adrenal chromaffin cells.

Dissociated bovine adrenal chromaffin cells in culture were utilized to study the mechanisms for development of cellular tolerance to ethanol. Three days after plating, cells were grown in either control medium or medium containing 200 mM ethanol for periods up to 6 days. Catecholamine release induced by carbachol, 80 mM K+, BAY K 8644, and veratrine was studied in ethanol-treated and untreated cultures. The presence of ethanol in vitro was inhibitory to all these stimuli in untreated cell cultures, carbachol-induced release being most sensitive (IC50 approximately 30 mM). After growth in ethanol for 6 days, carbachol-induced catecholamine release was completely resistant to the inhibitory effects of ethanol. The resistance to ethanol of carbachol-induced catecholamine release from cells grown in medium containing ethanol also extended to the inhibitory effects of butanol. The results show some similarities with changes observed in the central nervous system during the development of ethanol tolerance, and it is suggested that this model system may be useful in examining the mechanisms by which tolerance occurs.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Genetic up-regulation of calcium channels in a cellular model of ethanol dependence.

Bovine adrenal chromaffin cells were maintained in culture medium containing ethanol (200 mM) for 6 days. Cultures maintained in ethanol were viable and were morphologically similar to controls. There was a greater than 100% increase in the number of [3H]dihydropyridine calcium channel antagonist binding sites on the cell membranes from ethanol-treated cultures, which could be prevented by concomitant exposure to cycloheximide (5 micrograms.ml-1) and the mRNA synthesis inhibitor, anisomycin (10 micrograms.ml-1) implicating de novo synthesis of protein and genetic regulation, respectively. Release of catecholamines, induced by 18 mM K+, from cultures grown in ethanol was enhanced. The increased release of catecholamines was inhibited by nM concentrations of dihydropyridine calcium channel antagonists, implying that an increase in the number of dihydropyridine-sensitive calcium channels accounts for this functional alteration.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Altered characteristics of catecholamine release from rat cortical slices and bovine adrenal chromaffin cells in culture after chronic exposure to ethanol and the effect of the dihydropyridine drugs on these systems.

The depolarisation-induced release of dopamine from control rat striatal slices is inhibited by ethanol in vitro. The same parameter is enhanced in preparations from ethanol-treated rats. Under conditions of superfusion with normal physiological solutions we were unable to see similar results in noradrenaline (NA) release from cortical slices. Since we believed altered intracellular Ca2+ metabolism might underlie changes induced by ethanol in dopamine neurones, we superfused cortical slices with low K+ solutions which raise intracellular Ca2+. Under these conditions, ethanol in vitro inhibited depolarisation-induced NA release in control preparations. In preparations from ethanol tolerant animals, ethanol enhanced depolarisation-induced release. In cultures of adrenal chromaffin cells, similar alterations in coupling between Ca2+ and catecholamine release can be seen. Some of these changes may be a consequence of increased functional activity of dihydropyridine-sensitive Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1977

Influence of the root tip and the duration of washing on k retention by excised apical root segments of corn.

Excised apical segments of corn root (Zea mays) (5-15 mm from the root cap), some with and some without the root tips (0-5 mm) attached, were washed for varying time periods up to 4 hours in 0.5 mm CaSO(4). After washing, tips were removed from those segments washed with tips attached, and then all segments and tips were analyzed for K(+) content. The root tips (0-5 mm) initially contained about twice the K(+) of the apical segments (5-15 mm). The loss of K(+) did not exceed 15% in the tips or 20% in the apical segments. Loss of K(+) was most pronounced during the 1st hour of washing. There was little difference in K(+) content of apical segments washed with tips attached compared with those washed tipless. Thus, the presence of the intact root tip had no consistent influence on the ability of the older root tissue to retain K(+).

Journal Article↗