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The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions -287 to -263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.

Incidence of scrapie in sheep is strongly associated with PrP gene amino acid codon variants at positions 136, 154 and 171. However, there are breed differences in disease linkage and anomalous disease patterns which cannot obviously be explained by the '3 codon' genotype. Mouse studies indicate that PrP protein levels can influence scrapie disease progression and this prompted us to study the sheep PrP gene promoter region in a search for novel polymorphisms which may influence gene expression and hence disease susceptibility. The incidence of three single nucleotide polymorphisms (SNP) at positions C/A-5354, T/C-5382 and C/G-5622 within the PrP gene promoter region was determined from Neuropathogenesis Unit (NPU) and New Zealand (NZ) Cheviot and UK and NZ Suffolk sheep. The SNP variants A-5354 and G-5622 created consensus sequences for STAT and SP1 transcription factors, respectively, and C-5382 was within Motif 1, one of four conserved motifs found within the promoter region of mammalian PrP genes. The occurrence of C/A-5354 and T/C-5384 SNP exhibited differential associations with the PrP open reading frame (ORF) variants linked to scrapie susceptibility. A significant imbalance in the incidence of the C-5354/AXQ haplotype was found in the NPU Cheviot flock. C-5382 was not found in Suffolk sheep of either UK or NZ origin. The G-5622 SNP was found at a lower incidence in Suffolk sheep compared with Cheviots. The range of transcription factor binding motif profiles in the PrP gene promoter may act to modulate PrP gene activity and warrants further large-scale study.

One hundred and ninety nine cholescintigrams were performed at our hospital between January 1993 and December 1998. Of these, 96 were performed as part of the investigation of right upper quadrant or epigastric pain in patients whose prior biliary ultrasound scans had shown no abnormalities. In this group of patients, 60 cholescintigrams were reported as normal, 28 as abnormal and eight as equivocal. A review of the case notes for this group was made, with 91 out of 96 case notes being retrieved. Thirteen of the 58 (22%) patients with normal cholescintigrams either underwent or had been offered cholecystectomy, compared with 22 of the 27 (81%) with 'definitely abnormal' cholescintigrams. Of the subset of patients with abnormal cholescintigrams subsequently undergoing cholecystectomy, 16 out of 18 were found to have histologically abnormal gallbladders, while 11 out of 18 reportedly had their symptoms cured or improved. A fatty meal is used in our unit as the stimulus to gallbladder contraction, in contrast to the majority of papers published in the literature. We believe that the use of a fatty meal is acceptable and that the use of intravenous exogenous cholecystokinin is probably best reserved for the small group of patients with equivocal scintigrams who may require further investigation.

Cell-cycle phase of the donor and recipient cells at the moment of nuclear transfer influences subsequent development of the reconstituted embryo. In order to study this effect, the precise cell-cycle phase of the recipient oocyte at the time of fusion must be known and this depends on reliable activation of oocytes in a protocol that has a low incidence of spontaneous activation. Mouse oocytes recovered before (8-10 hours post-human chorionic gonadotropin [hCG]) and after ovulation (14 and 18 hours post-hCG) were exposed to strontium ions in calcium magnesium-free M16 culture medium. The effect on development of haploid parthenotes of post-hCG age of the oocyte, the duration of exposure, and strontium concentration in the medium was determined. These experiments established a reliable method of parthogenetic activation of recently ovulated mouse oocytes, involving the culture of oocytes for 60 minutes in 25 mM strontium in a calcium magnesium-free M16 medium. This method of activation was also able to induce activation of preovulatory oocytes after a preincubation period in vitro. Only a low incidence of spontaneous activation was observed if oocytes were recovered before or immediately after ovulation (14 hours after hCG).

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine beta-lactoglobulin-human alpha 1-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease DpnI, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos.

Mouse 2-, 4-, 8-, and 16-cell embryos were exposed to nocodazole in M16 culture medium. The effect of different concentrations and exposure times on the efficiency of cell cycle synchronization and the development of the treated embryos after release from the drug was determined. The minimum effective concentration (> 95% of arrested nuclei) for 4-, 8-, and 16-cell embryos was 5 microM nocodazole. The effect upon subsequent development of mouse embryos depended upon both the stage of development of the embryo at treatment (P < 0.001) and the length of exposure to nocodazole (P < 0.001). Exposure to any concentration of nocodazole within the range 2.5-10 microM for 12 hr caused a reduction in the proportion of embryos that formed blastocysts. As the period of exposure to 5 microM nocodazole increased from 12 to 24 hr, the proportion of embryos developing to the blastocyst stage decreased. The lower proportion of embryos developing to the blastocyst stage and to term (P < 0.01) suggests that the more advanced stages were more susceptible to damage as a result of exposure to nocodazole. The rate of development of 4-cell embryos to blastocysts was not affected when an exposure time of 9 hr was used. Together these results show that it is possible to use nocodazole to arrest mouse embryonic cells in mitosis but that it is not appropriate to culture the embryos in the presence of this drug for prolonged periods. Individual blastomeres completed mitosis at 60-90 min and started DNA synthesis at 120-150 min after release from nocodazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Micromanipulation techniques were used to produce reconstituted one-cell mouse embryos after the fusion of fetal male germ cells 15.5 day post coitum with enucleated secondary oocytes. At this stage of development, male fetal germ cells are arrested at G1 of mitotic interphase. Two distinct populations of germ cells, differing in size and ploidy, were isolated from the genital ridge of a mid-term fetus. Oocytes that had received male germ cells from the population of smaller (mononuclear) germ cells developed as diploid one-cell reconstituted embryos. When the same procedures were used to produce reconstituted one-cell embryos using male fetal germ cells from a population of larger (multinucleate) cells, they exhibited ploidy of either 4x, 6x or 8x at metaphase of the first cell division. Although most reconstituted embryos (90 and 96%) developed to the two-cell stage, the proportion of embryos receiving small germ cells developed to blastocysts was much higher (62%) than that receiving large germ cells (4%). These studies indicate that not all fetal germ cells are diploid before the onset of meiosis and have identified procedures to produce reconstituted embryos from fetal germ cells that do not carry genome or chromosome anomalies.

The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2-10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two-pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr(2+)-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr(2+)-induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.

Tetraploid mouse embryos were produced at the two-cell stage by blastomere fusion induced by inactivated Sendai virus. The embryos were from chromosomally normal female mice that had been fertilised by homozygous Rb(1.3)1Bnr males carrying a pair of large metacentric marker chromosomes in their karyotype. These "reconstructed" one-cell tetraploid embryos were then transferred to the oviducts of pseudopregnant recipients, which were subsequently autopsied early on the 10th day of gestation. Two-cell stage embryos that did not undergo blastomere fusion after 4-5 h were transferred to a second group of recipients, which were also autopsied early on the 10th day of gestation. From a total of 153 tetraploid embryos transferred to females that subsequently became pregnant, 135 implanted. Sixty-eight implantation sites were found to contain resorptions, whereas 67 contained mostly headfold presomite-stage embryos. Four embryos possessed four to six pairs of somites. All 57 embryos that could be analysed cytogenetically were found to be tetraploid. G-banding analysis revealed that 30 of these embryos had an XXYY and 27 and XXXX sex-chromosome constitution. The presence of two marker chromosomes in all mitotic preparations from each of these tetraploid embryos confirmed that they had all been produced by duplication of their original XY or XX diploid chromosome constitution, respectively. The XXYY:XXXX sex ratio observed was not significantly different from unity. In the control series of transfers, all of the embryos recovered were at the forelimb bud stage and had a diploid chromosome constitution. The results reported here differ from human clinical findings, in which the XXYY:XXXX sex ratio of 120 human tetraploid spontaneous abortions recovered over the last 20 years is 45:75. Possible explanations for these differences are briefly discussed.

In this study, the chromosome constitution of mouse eggs exposed in vivo to a dilute solution of ethanol during specific stages of the first and second meiotic divisions was determined at the first cleavage mitosis. Exposure to ethanol prior to the completion of the second meiotic division induced an incidence (7-10%) of aneuploidy involving only one chromosome in 98% of malsegregation events. This investigation provides indirect evidence that ethanol may induce aneuploidy by disrupting the functioning of the meiotic spindle. Karyological analyses of chromosome spreads prepared at the first cleavage metaphase suggest that only a small proportion of the total chromosome complement may be induced to undergo malsegregation.

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic "tetrad" chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL x CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromosome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited "homologous pairing," and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature "cytoplasmic maturation" of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.

The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.

A high incidence of parthenogenetic activation was observed when postovulatory aged mouse oocytes were exposed briefly to hyaluronidase in culture medium at 18-26 h after the human chorionic gonadotropin injection for inducing superovulation. The majority of the activated oocytes extruded a second polar body and developed a single haploid pronucleus. Cytogenetic analysis of this class of parthenogenone at metaphase of the first-cleavage mitosis has clearly demonstrated that the completion of the second meiotic division in activated aged oocytes is not associated with a significant increase in the incidence of chromosome segregation errors. The increasing postovulatory age of oocytes prior to activation was observed to significantly decrease the capacity of activated oocytes to extrude the second polar body.

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.

The brief exposure of recently ovulated mouse oocytes to a dilute solution of ethanol in vitro for 1, 3, or 5 min induced a uniform high incidence of parthenogenetic activation. The majority of parthenogenones developed a single haploid pronucleus after the extrusion of a second polar body. The proportionate incidence of this parthenogenetic class was significantly reduced as the duration of ethanol exposure increased from 1 min to 5 min. There was a concomitant increase in the incidence of parthenogenones that developed two haploid pronuclei following failure of extrusion of the second polar body. Cytogenetic analysis of the ethanol-induced single-pronuclear haploid parthenogenones at metaphase of the first cleavage division clearly demonstrated that a significant proportion were aneuploid. The incidence of aneuploidy observed was directly related to the duration of ethanol exposure. G-band analysis of the aneuploid metaphases revealed that the chromosomes were not randomly involved in the malsegregation events. This observation may be a reflection of the relationship of particular chromosomes to the meiotic spindle apparatus rather than on any specific property of the agent to which they were exposed. It is believed that ethanol disrupts the organisation of cytoskeletal elements and, in particular, interferes with the processes of chromosome segregation at the second meiotic division.